Nitrogen-containing macrocyclic conjugates as radiopharmaceuticals

ABSTRACT

The present invention relates to compounds that are useful as metal ligands and which either contain a molecular recognition moiety or can be bound to a molecular recognition moiety and methods of making these compounds. Once the compounds that contain a molecular recognition moiety are coordinated with a suitable metallic radionuclide, the coordinated compounds are useful as radiopharmaceuticals in the areas of radiotherapy and diagnostic imaging. The invention therefore also relates to methods of diagnosis and therapy utilising the radiolabelled compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 16/712,485, filed Dec. 12, 2019, which is a continuation of U.S.application Ser. No. 15/612,009, filed Jun. 2, 2017, now U.S. Pat. No.10,544,164, issued Jan. 28, 2020, which is a Continuation application ofU.S. application Ser. No. 13/132,194, filed on Aug. 22, 2011, now U.S.Pat. No. 9,701,694, issued on Jul. 11, 2017, which is a National StageApplication of PCT/AU2009/001572, filed Dec. 2, 2009, which claimspriority to Australian Application 2008906239, filed Dec. 2, 2008, thecontents of which are incorporated by reference herein in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03 (a)), a Sequence Listing in the form of anASCII-compliant text file (entitled“Sequence_listing_2913474-009005_ST25.txt” created on Jul. 21, 2021, and1,071 bytes in size) is submitted concurrently with the instantapplication, and the entire contents of the Sequence Listing areincorporated herein by reference.

FIELD

The present invention relates to compounds that are useful as metalligands and which either contain a molecular recognition moiety or canbe bound to a molecular recognition moiety and methods of making thesecompounds. Once the compounds that contain a molecular recognitionmoiety are coordinated with a suitable metallic radionuclide, thecoordinated compounds are useful as radiopharmaceuticals in the areas ofradiotherapy and diagnostic imaging. The invention therefore alsorelates to methods of diagnosis and therapy utilising the radiolabelledcompounds of the invention.

BACKGROUND

Radiolabelled compounds may be used as radiopharmaceuticals in a numberof applications such as in radiotherapy or diagnostic imaging. In orderfor a radiolabelled compound to be employed as a radiopharmaceuticalthere are a number of desirable properties that the compound shouldideally possess such as acceptable stability and, where possible, adegree of selectivity or targeting ability.

Initial work in the areas of radiopharmaceuticals focused on simplemetal ligands which were generally readily accessible and hence easy toproduce. A difficulty with many of these radiolabelled compounds is thatthe complex formed between the ligand and the metal ion was notsufficiently strong and so dissociation of the metal ion from the ligandoccurred in the physiological environment. This was undesirable as withthe use of ligands of this type there was no ability to deliver theradiopharmaceutical to the desired target area in the body as metalexchange with metal ions in the physiological environment meant thatwhen the radiopharmaceutical compound arrived at the desired site ofaction the level of radiolabelled metal ion coordinated to the compoundhad become significantly reduced. In addition where this type ofexchange is observed the side effects experienced by the subject of theradiotherapy or radioimaging are increased as radioactive material isdelivered to otherwise healthy tissue in the body rather thanpredominantly to its place of action.

In order to overcome the problem of metal dissociation in thephysiological environment a number of more complicated ligands have beendeveloped and studied over time. Thus, for example a wide range oftetra-azamacrocycles based on the cyclam and cyclen framework have beeninvestigated. Examples of ligands of this type include DOTA and TETA.

Unfortunately, even with these ligands there is still dissociation ofthe metal with certain derivatives. For example, some derivatives sufferfrom dissociation of Cu from the chelate as a consequence oftranschelation to biological ligands such as copper transport proteinseither as Cu²⁺ or following in vivo reduction to Cut

In order to increase the stability of radiolabelled compounds thereforehexaminemacrobicyclic cage amine ligands, known by their trivial namesarcophagines have been developed. These cage ligands form remarkablystable complexes with metals such as Cu²⁺ and have fast complexationkinetics even at low concentrations of metal at ambient temperatures.These features therefore make ligands of this type particularly wellsuited in radiopharmaceutical applications, especially thoseapplications involving copper.

Once the problem of stability of the complex between the ligand and themetal had been overcome attention turned to developing ways in which theligand could be functionalised to incorporate targeting molecules withinthe ligand without compromising the stability of the metal ligandcomplex or the ultimate biological activity of the targeting molecule. Anumber of different targeting molecules are known in the art and theissue became how best to attach these to the ligand molecules.

In general the targeting molecule (or molecular recognition moiety as itis sometimes known) is attached to the ligand to provide a finalcompound containing both a ligand and a molecular recognition moiety.Whilst these compounds may contain a single molecular recognition moietythey may also be multimeric constructs where the ligand is attached totwo (or more) molecular recognition moieties. This is typicallydesirable as a multimeric construct can possess higher affinity for atarget receptor than its monomeric equivalent. This is in part due to anincrease in the local concentration of the targeting group, allowing itto compete more effectively with endogenous ligands. In addition incircumstances where there is sufficient length between two or moretargeting groups within a multimeric construct, then cooperative bindingis possible, and two or more targeting groups will bind to two or morereceptor sites at the same time. Indeed it has been observed that invivo, a multimeric construct often demonstrates higher target tissueaccumulation than its monomeric equivalent. Without wishing to be boundby theory it is thought that this is due to the higher affinity of themultimeric construct for the target receptor than that of the monomericconstruct. Furthermore, the multimeric construct has a higher molecularweight than the monomeric construct and therefore prolongedbioavailability (as it is more resistant to degradation in thephysiological environment). This can result in increased accumulationand retention in target tissue.

Initial work in the caged ligand area looked at direct couplingreactions of the primary amines of the cage amine ‘diaminosarcophagine’,1,8-diamino-3,6,10,13,16,19-hexaaza bicyclo[6.6.6] icosane ((NH₂)₂sar),with peptides using standard coupling procedures. Unfortunately for avariety of reasons this has proven to be relatively inefficient and workin this area ceased. Workers then focussed on the incorporation of anaromatic amine to produce SarAr. The pendent aromatic amine can be usedin conjugation reactions with the carboxylate residues of peptides orantibodies and it has been shown that SarAr could be conjugated toanti-GD2 monoclonal antibody (14.G2a) and its chimeric derivative(ch14.8) and the conjugate has been radiolabelled with ⁶⁴Cu.

A difficulty with this approach is that in reaction of the aromaticamine in the conjugation step there are 8 other nitrogen atoms in theSarAr molecule that are available for competing reactions leading to thepotential for the creation of a large number of impurities that isundesirable from a pharmaceutical sense. Whilst these could potentiallybe overcome by the use of substantial protective group chemistry this isclearly undesirable from a synthetic standpoint and scale up on acommercial scale.

An alternative approach has been to elaborate the ligand to incorporatecarboxylate functional groups and incorporate peptides or antibodies viatheir N-terminal amine residues and this approach is of particularimportance when the C-terminus is crucial to biological activity.Studies have shown that (NH₂)₂sar, can be functionalised with up to fourcarboxymethyl substituents via alkylation reactions with chloroaceticacid and the introduced carboxymethyl arms can be used as a point offurther functionalisation and EDC-coupling reactions can then be used tointroduce amino acids.

Unfortunately a potential disadvantage of these systems is thatintramolecular cyclisation reactions can still occur in which thecarboxymethyl arm reacts with a secondary amine of the cage framework toform lactam rings resulting in quadridentate rather than sexidentateligands. Accordingly whilst this approach can be followed the potentialfor unwanted side reactions is clearly undesirable from a commercialperspective.

Accordingly there is still a need to develop compounds that are capableof being radiolabelled and which contain, or are capable of being boundto a molecular recognition moiety that can be used inradiopharmaceutical applications. In addition it would be desirable ifthe compounds had the flexibility to provide for the possibility ofmultimeric constructs being used as in certain circumstances theseconstructs demonstrate higher biological activity as discussed above.

SUMMARY

In one aspect there is provided a compound of formula (I),

wherein:

L is a nitrogen containing macrocyclic metal ligand;

X is a linking moiety;

Y is selected from the group consisting of OR, SR¹ and N(R²)₂;

R is independently selected from the group consisting of H, an oxygenprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

R¹ is independently selected from the group consisting of H, a sulfurprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

each R² is independently selected from the group consisting of H, anitrogen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl;

or a pharmaceutically acceptable salt or complex thereof.

In a further aspect there is provided a compound of formula (II),

wherein:

L is a nitrogen containing macrocyclic metal ligand;

X is a linking moiety;

Y is a molecular recognition moiety;

or a pharmaceutically acceptable salt or complex thereof.

As with any group of structurally related compounds which possess aparticular utility, certain embodiments of variables of the compounds ofthe formula (I) and formula (II), which are particularly useful in theirend use application.

In the compounds of formula (I) and formula (II) the X moiety serves asa linking moiety that serves to act as a spacer between the two carbonylmoieties which separate the ligand which can be bound to theradionuclide and either the point of attachment of a molecularrecognition moiety or the molecular recognition moiety per se. As suchwhilst it is desirable that there be a certain degree of separationbetween the two in order to ensure that the two entities do notinterfere with each other's activity it is also important that the twoare not so far removed such that the radionuclide is not effectivelydelivered to its site of operation.

In some embodiments X is a linking moiety having from 1 to 20 atoms inthe normal chain. In some embodiments X is a linking moiety having from1 to 15 atoms in the normal chain. In some embodiments X is a linkingmoiety having from 1 to 12 atoms in the normal chain. In someembodiments X is a linking moiety having from 1 to 10 atoms in thenormal chain. In some embodiments X is a linking moiety having from 1 to8 atoms in the normal chain. In some embodiments X has 8 atoms in thenormal chain. In some embodiments X has 7 atoms in the normal chain. Insome embodiments X has 6 atoms in the normal chain. In some embodimentsX has 5 atoms in the normal chain. In some embodiments X has 4 atoms inthe normal chain. In some embodiments X has 3 atoms in the normal chain.In some embodiments X has 2 atoms in the normal chain. In someembodiments X has 1 atom in the normal chain.

A wide range of possible moieties may be use to create a linking moietyof this type. Examples of suitable moieties that may be used in thecreation of X include optionally substituted C₁-C₁₂alkyl, substitutedC₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl.

In some embodiments X is a group of the formula:

—(CH₂)_(q)CO(AA)_(r)NH(CH₂)_(s)—

wherein each AA is independently an amino acid group;

q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

r is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8, and (AA)_(r) is SEQ ID NO:3; and

s is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8.

In some embodiments q is 1. In some embodiments q is 2. In someembodiments q is 3. In some embodiments q is 4. In some embodiments q is5. In some embodiments q is 6. In some embodiments q is 7. In someembodiments q is 8.

In some embodiments r is 0. In some embodiments r is 1. In someembodiments r is 2. In some embodiments r is 3. In some embodiments r is4. In some embodiments r is 5. In some embodiments r is 6. In someembodiments r is 7. In some embodiments r is 8.

In some embodiments s is 0. In some embodiments s is 1. In someembodiments s is 2. In some embodiments s is 3. In some embodiments s is4. In some embodiments s is 5. In some embodiments s is 6. In someembodiments s is 7. In some embodiments s is 8.

In some embodiments the amino acid is a naturally occurring amino acid.In some embodiments the amino acid is a non-naturally occurring aminoacid. In some embodiments the amino acid is selected from the groupconsisting of phenyl alanine, tyrosine, amino hexanoic acid andcysteine.

In some embodiments q is 3, r is o and s is 5. In these embodiments X isa group of the formula:

—(CH₂)₃CONH(CH₂)₅—

In some embodiments X is a group of the formula:

—(CH₂)_(n)—,

wherein optionally one or more of the CH₂ groups may be independentlyreplaced by a heteroatomic group selected from S, O, P and NR³ where R³is selected from the group consisting of H, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl; and

n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9 and 10.

In some embodiments n is selected from the group consisting of 1, 2, 3,4, and 5. In some embodiments n is 4. In some embodiments n is 3. Insome embodiments n is 3. In some embodiments n is 1.

In some embodiments X is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.

In some embodiments X is —(CH₂)—. In some embodiments X is —(CH₂)₂—. Insome embodiments X is —(CH₂)₃—. In some embodiments X is —(CH₂)₄—. Insome embodiments X is —(CH₂)₅—. In some embodiments X is —(CH₂)₆—. Insome embodiments X is —(CH₂)₇—. In some embodiments X is —(CH₂)₈—. Insome embodiments X is —(CH₂)₉—. In some embodiments X is —(CH₂)₁₀—.

The compounds of formula (I) and formula (II) may include any of anumber of nitrogen containing macrocyclic metal ligands.

In some embodiments the ligand (L) may be a tetra-azamacrocycle based onthe cyclam and cyclen framework. In some embodiments L is a nitrogencontaining cage metal ligand. Cage ligands of this type are typicallyuseful as they bind strongly to metal ions leading to a stable complexbeing formed.

In some embodiments L is a nitrogen containing cage metal ligand of theformula:

V is selected from the group consisting of N and CR⁴;

each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO;

each p is independently an integer selected from the group consisting of2, 3, and 4;

R⁴ is selected from the group consisting of H, OH, halogen, NO₂, NH₂,optionally substituted C₁-C₁₂alkyl, optionally substituted C₆-C₁₈aryl,cyano, CO₂R⁵, NHR⁵, N(R⁵)₂ and a group of the formula:

wherein:

X¹ is a linking moiety;

Y¹ is selected from the group consisting of OR⁶, SR⁷, N(R⁸)₂ and amolecular recognition moiety;

wherein R⁵ is H or C₁-C₁₂alkyl.

R⁶ is selected from the group consisting of H, halogen, an oxygenprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

R⁷ is selected from the group consisting of H, halogen, a sulfurprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

each R⁸ is independently selected from the group consisting of H, anitrogen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl.

In some embodiments L is a macrocyclic metal ligand of the formula:

wherein R^(x), R^(y) and p are as defined above.

In some embodiments L is a macrocyclic ligand of the formula:

wherein R^(x), R^(y), R⁴ and p are as defined above.

In some embodiments L is selected from the group consisting of:

In some of the embodiments of the ligand L, the ligand is furtherfunctionalised or substituted by a group R⁴. This allows for theformation of bi-functional ligands as there is the potential for thegroup R⁴ to complement the existing functionality of the ligand or toprovide additional functionality if required. In addition where one istrying to make a multimeric construct the group R⁴ is typically thegroup used to introduce the second targeting or molecular recognitionmoiety.

In some embodiments R⁴ is selected from the group consisting of NH₂, CH₃and a group of the formula:

X¹ is a linking moiety;

Y¹ is selected from the group consisting of OR⁶, SR⁷, N(R⁸)₂ and amolecular recognition moiety;

R⁶ is selected from the group consisting of H, halogen, an oxygenprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

R⁷ is selected from the group consisting of H, halogen, a sulfurprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

each R⁸ is independently selected from the group consisting of H, anitrogen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl.

In some embodiments X¹ is a linking moiety having from 1 to 20 atoms inthe normal chain. In some embodiments X¹ is a linking moiety having from1 to 15 atoms in the normal chain. In some embodiments X¹ is a linkingmoiety having from 1 to 12 atoms in the normal chain. In someembodiments X¹ is a linking moiety having from 1 to 10 atoms in thenormal chain. In some embodiments X¹ is a linking moiety having from 1to 8 atoms in the normal chain. In some embodiments X¹ has 8 atoms inthe normal chain. In some embodiments X¹ has 7 atoms in the normalchain. In some embodiments X¹ has 6 atoms in the normal chain. In someembodiments X¹ has 5 atoms in the normal chain. In some embodiments X¹has 4 atoms in the normal chain. In some embodiments X¹ has 3 atoms inthe normal chain. In some embodiments X¹ has 2 atoms in the normalchain. In some embodiments X¹ has 1 atom in the normal chain.

A wide range of possible moieties may be use to create a linking moietyof this type. Examples of suitable moieties that may be used in thecreation of X¹ include optionally substituted C₁-C₁₂alkyl, substitutedC₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl.

In some embodiments X¹ is a group of the formula:

—(CH₂)_(t)CO(AA)_(u)NH(CH₂)_(v)—

wherein each AA is independently an amino acid group;

t is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

u is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8, and (AA)_(u) is SEQ ID NO:3; and

v is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8.

In some embodiments t is 1. In some embodiments t is 2. In someembodiments t is 3. In some embodiments t is 4. In some embodiments t is5. In some embodiments t is 6. In some embodiments t is 7. In someembodiments t is 8.

In some embodiments u is 0. In some embodiments u is 1. In someembodiments u is 2. In some embodiments u is 3. In some embodiments u is4. In some embodiments u is 5. In some embodiments u is 6. In someembodiments u is 7. In some embodiments u is 8.

In some embodiments v is 0. In some embodiments v is 1. In someembodiments v is 2. In some embodiments v is 3. In some embodiments v is4. In some embodiments v is 5. In some embodiments v is 6. In someembodiments v is 7. In some embodiments v is 8.

In some embodiments the amino acid is a naturally occurring amino acid.In some embodiments the amino acid is a non-naturally occurring aminoacid. In some embodiments the amino acid is selected from the groupconsisting of phenyl alanine, tyrosine, amino hexanoic acid andcysteine.

In some embodiments t is 3, u is o and v is 5. In these embodiments X¹is a group of the formula:

—(CH₂)₃CONH(CH₂)₅—

In some embodiments X¹ is a group of the formula

—(CH₂)_(a)—,

wherein optionally one or more of the CH₂ groups may be independentlyreplaced by a heteroatomic group selected from S, O, P and NR⁹ where R⁹is selected from the group consisting of H, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl;

a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9 and 10.

In some embodiments a is selected from the group consisting of 1, 2, 3,4, and 5. In some embodiments a is 4. In some embodiments n is 3. Insome embodiments a is 2. In some embodiments a is 1.

In some embodiments X¹ is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.

In some embodiments X¹ is —(CH₂)—. In some embodiments X¹ is —(CH₂)₂—.In some embodiments X¹ is —(CH₂)₃—. In some embodiments X¹ is —(CH₂)₄—.In some embodiments X¹ is —(CH₂)₅—. In some embodiments X¹ is —(CH₂)₆—.In some embodiments X¹ is —(CH₂)₇—. In some embodiments X¹ is —(CH₂)₈—.In some embodiments X1 is —(CH₂)₉—. In some embodiments X¹ is —(CH₂)₁₉—.

In some embodiments Y¹ is OH or a molecular recognition moiety. In someembodiments Y¹ is OH. In some embodiments Y¹ is a molecular recognitionmoiety.

In some embodiments L is a group of the formula:

In some embodiments of the compounds of formula (I) Y is OH.

In the compounds of formula (II) Y is a molecular recognition moiety. Insome embodiments of the compounds of both formula (I) and formula (II)Y¹ is a molecular recognition moiety.

In those embodiments where Y or Y¹ are a molecular recognition moiety itmay be any moiety that has the ability to recognise a target moiety in aphysiological environment. In some embodiments the molecular recognitionmoiety includes a molecular recognition portion which is directlyattached to the remainder of the molecule. In some embodiments themolecular recognition moiety includes a spacer portion and a molecularrecognition portion wherein the spacer portion joins the molecularrecognition portion to the remainder of the molecule. The spacer may beany suitable construct and is typically chosen such that it provides asuitable distance or “space” between the ligand moiety of the moleculeand the molecular recognition portion of the molecule. The exact length(if required at all) will vary depending upon the specific targetreceptor, the nature of the ligand and the nature of the molecularrecognition portion. In some instances the spacer may be desirable as itallows for more facile synthesis of the compounds of the invention basedon the nature of the molecular recognition portion.

In some embodiments the molecular recognition moiety or molecularrecognition portion is selected from the group consisting of anantibody, a protein, a peptide, a carbohydrate, a nucleic acid, anoligonucleotide, an oligosaccharide and a liposome or a fragment orderivative thereof.

In some embodiments the molecular recognition moiety or molecularrecognition portion is an antibody or a fragment or derivative thereof.In some embodiments the molecular recognition moiety is a protein or afragment or derivative thereof. In some embodiments the molecularrecognition moiety or molecular recognition portion is a peptide or afragment or derivative thereof. In some embodiments the molecularrecognition moiety or molecular recognition portion is a carbohydrate ora fragment or derivative thereof. In some embodiments the molecularrecognition moiety or molecular recognition portion is a nucleic acid ora fragment or derivative thereof. In some embodiments the molecularrecognition moiety or molecular recognition portion is anoligonucleotide or a fragment or derivative thereof. In some embodimentsthe molecular recognition moiety or molecular recognition portion is anoligosaccharide or a fragment or derivative thereof. In some embodimentsthe molecular recognition moiety or molecular recognition portion isfolic acid or a fragment or derivative thereof. In some embodiments themolecular recognition moiety or molecular recognition portion is vitaminB12 or a fragment or a derivative thereof. In some embodiments themolecular recognition moiety or molecular recognition portion is aliposome or a fragment or a derivative thereof.

In some embodiments the molecular recognition moiety or molecularrecognition portion is selected from the group consisting of Octreotate,octreotide, [Tyr³]-octreotate, [Tyr¹]-octreotate, bombesin,bombesin(7-14), gastrin releasing peptide, single amino acids,penetratin, annexin V, TAT, cyclic RGD, glucose, glucosamine (andextended carbohydrates), folic acid, neurotensin, neuropeptide Y,cholecystokinin (CCK) analogues, vasoactive intestinal peptide (VIP),substance P, and alpha-melanocyte-stimulating hormone (MSH).

In some embodiments the molecular recognition moiety or molecularrecognition portion is selected from the group consisting of[Tyr³]-octreotate and bombesin. In some embodiments the molecularrecognition moiety is [Tyr³]-octreotate. In some embodiments themolecular recognition moiety is Lys³-bombesin.

In some embodiments the molecular recognition moiety is cyclic RGD.

In some embodiments of the compounds of formula (I) or formula (II) thenitrogen containing macrocyclic metal ligand is complexed with a metalion. The ligand may be complexed with any suitable metal ion and may beused to deliver a range of metal ions. In some embodiments the metal ionis selected from the group consisting of Cu, Tc, Gd, Ga, In, Co, Re, Fe,Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg, andTi.

In some embodiments the metal ion is a radionuclide selected from thegroup consisting of Cu, Tc, Ga, Co, In, Fe, and Ti. The presentcompounds have been found to be particularly applicable useful inbinding copper ions. In some embodiments the metal ion is a radionuclideselected from the group consisting of ⁶⁰Cu, ⁶²Cu, ⁶⁴Cu and ⁶⁷Cu. In someembodiments the metal ion is ⁶⁰Cu. In some embodiments the metal ion is⁶²Cu. In some embodiments the metal ion is ⁶⁴Cu. In some embodiments themetal ion is ⁶⁷Cu.

The invention also relates to pharmaceutical compositions including acompound of the invention as described above and a pharmaceuticallyacceptable carrier, diluent or excipient.

In a further aspect there is provided method of producing a compound offormula (I),

wherein L, X and Y are as defined above; the method including

(a) reacting an amino substituted metal chelating ligand or a metalcomplex thereof of the formula:

L-NH₂

wherein L is a nitrogen containing macrocyclic metal ligand;

with an activated dicarbonyl compound; and

(b) isolating the compound of formula (I) or a metal complex thereof.

The activated dicarbonyl compound used in the synthetic methods of thepresent invention may be any suitable dicarbonyl compound. In someembodiments the activated dicarbonyl compound is a compound of theformula (III):

wherein X is as defined above and Z is O, S or NR².

In some embodiments the activated dicarbonyl compound is a compound ofthe formula (IV):

wherein X and Y are as defined above and L_(v) is a leaving group.

The leaving group may be any suitable group that can be displaced by thedesired incoming chemical moiety and a number of suitable leaving groupsare well known in the art. In some embodiments the leaving group isselected from the group consisting of Cl, Br, CH₃SO₃, CH₃C₆H₄SO₃, and agroup of the formula:

The compounds may be reacted under a wide variety of reaction conditionssuitable to facilitate the reaction. In some embodiments the aminosubstituted metal chelating ligand and the activated dicarbonyl compoundare reacted in the presence of a base. A number of suitable bases may beused. In some embodiments the base is diisopropylethylamine.

In yet a further aspect there is provided a method of treating orpreventing a condition in a subject, the method including the step ofadministering a therapeutically effective amount of a compound offormula (II) which is coordinated to a radionuclide to a subject. Insome embodiments the condition is cancer.

In yet a further aspect there is provided a method of radioimaging asubject, the method including the step of administering an effectiveamount of a compound of formula (II) which is coordinated to aradionuclide to a subject.

These and other features of the present teachings are set forth herein.

DETAILED DESCRIPTION

In this specification a number of terms are used which are well known toa skilled addressee. Nevertheless for the purposes of clarity a numberof terms will be defined.

As used herein, the term “unsubstituted” means that there is nosubstituent or that the only substituents are hydrogen.

The term “optionally substituted” as used throughout the specificationdenotes that the group may or may not be further substituted or fused(so as to form a condensed polycyclic system), with one or morenon-hydrogen substituent groups. In certain embodiments the substituentgroups are one or more groups independently selected from the groupconsisting of halogen, ═O, ═S, —CN, —NO₂, —CF₃, —OCF₃, alkyl, alkenyl,alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl,cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl,cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl,heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl,arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy,alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl,alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl,alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy,heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy,heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl,arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl,arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl,aminosulfinylaminoalkyl, —C(═O)OH, —C(═O)R^(a), —C(═O)OR^(a),C(═O)NR^(a)R^(b), C(═NOH)R^(a), C(═NR^(a))NR^(b)R^(c), NR^(a)R^(b),NR^(a)C(═O)R^(b), NR^(a)C(═O)OR^(b), NR^(a)C(═O)NR^(b)R^(c),NR^(a)C(═NR^(b))NR^(c)R^(d), NR^(a)SO₂R^(b), —SR^(a), SO₂NR^(a)R^(b),—OR^(a), OC(═O)NR^(a)R^(b), OC(═O)R^(a) and acyl,

wherein R^(a), R^(b), R^(c) and R^(d) are each independently selectedfrom the group consisting of H, C₁-C₁₂alkyl, C₁-C₁₂haloalkyl,C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-C₁₀ heteroalkyl, C₃-C₁₂cycloalkyl,C₃-C₁₂cycloalkenyl, C₂-C₁₂heterocycloalkyl, C₂-C₁₂ heterocycloalkenyl,C₆-C₁₈aryl, C₁-C₁₈heteroaryl, and acyl, or any two or more of R^(a),R^(b), R^(c) and R^(d), when taken together with the atoms to which theyare attached form a heterocyclic ring system with 3 to 12 ring atoms.

In some embodiments each optional substituent is independently selectedfrom the group consisting of: halogen, ═O, ═S, —CN, —NO₂, —CF₃, —OCF₃,alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy,alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy,cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy,heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl,heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl,arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl,aminoalkyl, —COOH, —SH, and acyl.

Examples of particularly suitable optional substituents include F, Cl,Br, I, CH₃, CH₂CH₃, OH, OCH₃, CF₃, OCF₃, NO₂, NH₂, and CN.

As used herein the term “amino acid” refers to a molecule which containsboth an amine and a carboxyl function. The amino acid may be a naturalor an unnatural amino acid.

“Alkenyl” as a group or part of a group denotes an aliphatic hydrocarbongroup containing at least one carbon-carbon double bond and which may bestraight or branched preferably having 2-12 carbon atoms, morepreferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in thenormal chain. The group may contain a plurality of double bonds in thenormal chain and the orientation about each is independently E or Z.Exemplary alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. Thegroup may be a terminal group or a bridging group.

“Alkyl” as a group or part of a group refers to a straight or branchedaliphatic hydrocarbon group, preferably a C₁-C₁₂ alkyl, more preferablya C₁-C₁₀ alkyl, most preferably C₁-C₆ unless otherwise noted. Examplesof suitable straight and branched C₁-C₆ alkyl substituents includemethyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl,and the like. The group may be a terminal group or a bridging group.

“Alkynyl” as a group or part of a group means an aliphatic hydrocarbongroup containing a carbon-carbon triple bond and which may be straightor branched preferably having from 2-12 carbon atoms, more preferably2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.Exemplary structures include, but are not limited to, ethynyl andpropynyl. The group may be a terminal group or a bridging group.

“Aryl” as a group or part of a group denotes (i) an optionallysubstituted monocyclic, or fused polycyclic, aromatic carbocycle (ringstructure having ring atoms that are all carbon) preferably having from5 to 12 atoms per ring. Examples of aryl groups include phenyl,naphthyl, and the like; (ii) an optionally substituted partiallysaturated bicyclic aromatic carbocyclic moiety in which a phenyl and aC₅₋₇ cycloalkyl or C₅₋₇ cycloalkenyl group are fused together to form acyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. Thegroup may be a terminal group or a bridging group. Typically an arylgroup is a C₆-C₁₈ aryl group.

“Cycloalkyl” refers to a saturated monocyclic or fused or spiropolycyclic, carbocycle preferably containing from 3 to 9 carbons perring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and thelike, unless otherwise specified. It includes monocyclic systems such ascyclopropyl and cyclohexyl, bicyclic systems such as decalin, andpolycyclic systems such as adamantane. A cycloalkyl group typically is aC₃-C₉ cycloalkyl group. The group may be a terminal group or a bridginggroup.

“Halogen” represents chlorine, fluorine, bromine or iodine.

“Heteroalkyl” refers to a straight- or branched-chain alkyl grouppreferably having from 2 to 12 carbons, more preferably 2 to 6 carbonsin the chain, in which one or more of the carbon atoms (and anyassociated hydrogen atoms) are each independently replaced by aheteroatomic group selected from S, O, P and NR′ where R′ is selectedfrom the group consisting of H, optionally substituted C₁-C₁₂alkyl,optionally substituted C₃-C₁₂cycloalkyl, optionally substitutedC₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl. Exemplaryheteroalkyls include alkyl ethers, secondary and tertiary alkyl amines,amides, alkyl sulfides, and the like. Examples of heteroalkyl alsoinclude hydroxyC₁-C₆alkyl, C₁-C₆alkyloxyC₁-C₆alkyl, aminoC₁-C₆alkyl,C₁-C₆alkylaminoC1-C₆alkyl, and di(C₁-C₆alkyl)aminoC₁-C₆alkyl. The groupmay be a terminal group or a bridging group.

“Heteroaryl” either alone or part of a group refers to groups containingan aromatic ring (preferably a 5 or 6 membered aromatic ring) having oneor more heteroatoms as ring atoms in the aromatic ring with theremainder of the ring atoms being carbon atoms. Suitable heteroatomsinclude nitrogen, oxygen and sulphur. Examples of heteroaryl includethiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole,benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan,isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole,pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole,isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine,naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine,acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole,isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-,or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3-indolyl, and2-, or 3-thienyl. A heteroaryl group is typically a C₁-C₁₈ heteroarylgroup. The group may be a terminal group or a bridging group.

A “leaving group” is a chemical group that is readily displaced by adesired incoming chemical moiety. Accordingly in any situation thechoice of leaving group will depend upon the ability of the particulargroup to be displaced by the incoming chemical moiety. Suitable leavinggroups are well known in the art, see for example “Advanced OrganicChemistry” Jerry March 4^(th) Edn. pp 351-357, Oak Wick and Sons NY(1997). Examples of suitable leaving groups include, but are not limitedto, halogen, alkoxy (such as ethoxy, methoxy), sulphonyloxy, optionallysubstituted arylsulfonyl. Specific examples include chloro, iodo, bromo,fluoro, ethoxy, methoxy, methansulphonyl, triflate and the like.

The term “normal chain” refers to the direct chain joining the two endsof a linking moiety.

The term “pharmaceutically acceptable salts” refers to salts that retainthe desired biological activity of the above-identified compounds, andinclude pharmaceutically acceptable acid addition salts and baseaddition salts. Suitable pharmaceutically acceptable acid addition saltsof compounds of Formula (I) may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids are hydrochloric,sulfuric, and phosphoric acid. Appropriate organic acids may be selectedfrom aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic andsulfonic classes of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Additionalinformation on pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co.,Easton, Pa. 1995. In the case of agents that are solids, it isunderstood by those skilled in the art that the inventive compounds,agents and salts may exist in different crystalline or polymorphicforms, all of which are intended to be within the scope of the presentinvention and specified formulae.

The term “therapeutically effective amount” or “effective amount” is anamount sufficient to effect beneficial or desired clinical results. Aneffective amount can be administered in one or more administrations. Aneffective amount is typically sufficient to palliate, ameliorate,stabilize, reverse, slow or delay the progression of the disease state.An effective amount for radioimaging is typically sufficient to identifythe radionuclide in the subject.

The term “molecular recognition moiety” refers to an entity capable ofbinding to a particular molecular entity, typically a receptor locationin the physiological environment. The term includes antibodies,proteins, peptides, carbohydrates, nucleic acids, oligonucleotides,oligosaccharides and liposomes.

The term “oxygen protecting group” means a group that can prevent theoxygen moiety reacting during further derivatisation of the protectedcompound and which can be readily removed when desired. In oneembodiment the protecting group is removable in the physiological stateby natural metabolic processes. Examples of oxygen protecting groupsinclude acyl groups (such as acetyl), ethers (such as methoxy methylether (MOM), β-methoxy ethoxy methyl ether (MEM), p-methoxy benzyl ether(PMB), methylthio methyl ether, Pivaloyl (Piv), Tetrahydropyran (THP)),andsilyl ethers (such as Trimethylsilyl (TMS) tert-butyl dimethyl silyl(TBDMS) and triisopropylsilyl (TIPS).

The term “nitrogen protecting group” means a group that can prevent thenitrogen moiety reacting during further derivatisation of the protectedcompound and which can be readily removed when desired. In oneembodiment the protecting group is removable in the physiological stateby natural metabolic processes and in essence the protected compound isacting as a prodrug for the active unprotected species. Examples ofsuitable nitrogen protecting groups that may be used include formyl,trityl, phthalimido, acetyl, trichloroacetyl, chloroacetyl, bromoacetyl,iodoacetyl; urethane-type blocking groups such as benzyloxycarbonyl(‘CBz’), 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, t-butoxycarbonyl(‘tBoc’), 2-(4-xenyl)-isopropoxycarbonyl,1,1-diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl,2-phenylprop-2-yloxycarbonyl, 2-(p-toluyl)-prop-2-yloxy-carbonyl,cyclo-pentanyloxy-carbonyl, 1-methylcyclopentanyloxycarbonyl,cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycarbonyl,2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfono)-ethoxycarbonyl,2-(methylsulfono)ethoxycarbonyl, 2-(triphenylphosphino)-ethoxycarbonyl,fluorenylmethoxycarbonyl (“FMOC”), 2-(trimethylsilyl)ethoxycarbonyl,allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalymethoxy carbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decycloxy)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonlyl and the like;benzoylmethylsulfono group, 2-nitrophenylsulfenyl, diphenylphosphineoxide, and the like. The actual nitrogen protecting group employed isnot critical so long as the derivatised nitrogen group is stable to thecondition of subsequent reaction(s) and can be selectively removed asrequired without substantially disrupting the remainder of the moleculeincluding any other nitrogen protecting group(s). Further examples ofthese groups are found in: Greene, T. W. and Wuts, P. G. M., ProtectiveGroups in Organic Synthesis, Second edition; Wiley-Interscience: 1991;Chapter 7; McOmie, J. F. W. (ed.), Protective Groups in OrganicChemistry, Plenum Press, 1973; and Kocienski, P. J., Protecting Groups,Second Edition, Theime Medical Pub., 2000.

The compounds of the invention as discussed above may include a widevariety of nitrogen containing macrocyclic metal ligands.

The ligand may be a monocyclic nitrogen containing metal ligand based onthe cyclam or cyclen frameworks. Ligand of this type and derivativesthereof may be synthesised using methodology available in the art suchas in Bernhardt (J. Chem. Soc., Dalton Transactions, 1996, pages4319-4324), Bernhardt et al (J. Chem. Soc., Dalton Transactions, 1996,pages 4325-4330), and Bernhardt and Sharpe (Inorg Chem, 2000, 39, pages2020-2025). Various other ligands of this general type may be made byvariation of the procedures described in these articles.

The ligand may also be a cage like cryptand ligand as described forexample in Geue (Chemical communications, 1994, page 667). Cryptandligands of this type are described in U.S. Pat. No. 4,497,737 in thename of Sargeson et al, the disclosure of which is incorporated hereinby reference.

The synthesis involves a metal ion template reaction and involvescondensation of a tris-(diamine) metal ion complex (see column 3 lines30 to 35) with formaldehyde and an appropriate nucleophile in thepresence of base. The identity of the nucleophile will determine theidentity of the substituents on the cage ligand and a skilled addresseecan access a wide variety of substitution patterns around the cageligand by judicious choice of the appropriate amine used in thecondensation as well as the identity of the nucleophile.

In order to produce the compounds of formula (I) of the invention theamino substituted ligand or a metal complexed form thereof is reactedwith an appropriate dicarbonyl compound under suitable reactionconditions to arrive at the final product.

Whilst the reaction may be performed on the free ligand there is still apossibility of the reaction being compromised by the presence of thering nitrogen(s). As such it is desirable to perform the reaction usinga metal complex thereof as the metal serves to act as a protecting groupfor the secondary nitrogen atoms in the ring.

The reaction may be carried out in any suitable solvent which is inertto the two reactants with the identity of the solvent being determinedby the relative solubilities of the anhydride and the amine substitutedmetal ligand. Examples of solvents that may be used include aliphatic,aromatic, or halogenated hydrocarbons such as benzene, toluene, xylenes;chlorobenzene, chloroform, methylene chloride, ethylene chloride; ethersand ethereal compounds such as dialkyl ether, ethylene glycol monoor-dialkyl ether, THF, dioxane; nitriles such as acetonitrile or2-methoxypropionitrile; N, N-dialkylated amides such asdimethylformamide; and dimethyl acetamide, dimethylsulphoxide,tetramethylurea; as well as mixtures of these solvents with each other.

The reaction may be carried out at any of a number of suitabletemperatures with the reaction temperature being able to be readilydetermined on a case by case basis. Nevertheless the reactiontemperature is typically carried out at from 0 to 100° C., moretypically 50 to 80° C.

The reaction may be carried out using a wide variety of activateddicarbonyl compounds. In some embodiments the activated dicarbonylcompound is an anhydride of the formula:

wherein X is as defined above and Z is O, S or NR².

Anhydride compounds of this type are generally readily available forcertain values of X and thus these compounds may be readily used forvalues of X for which they are obtainable. It is desirable that they beutilised where possible as the potential for side reactions is reducedsomewhat with these compounds.

In some embodiments the activated dicarbonyl compound is a compound ofthe formula:

wherein X and Y are as defined above and L_(v) is a leaving group. TheL_(v) group on the compounds of this type may be any suitable leavinggroup but is typically selected from the group consisting of Cl, Br,CH₃SO₃, CH₃C₆H₄SO₃, and a group of the formula:

In choosing a suitable leaving group for reactions of this type theskilled worker in the art will have regard for the functionality of theremainder of the molecule and the ease of production of the activateddicarbonyl compound in each instance.

The reaction is also typically carried out in the presence of a base asthis is found to facilitate the reaction. Examples of suitable basesinclude hindered tertiary amines with trialkyl amines such astrimethylamine, triethyleneamine, diisopropylethyl amine being suitableexamples of bases for use in the reaction. The amount of base used issuch that it is in a significant molar excess so as to ensure that thereaction does not become affected by acidification as it progresses.

The exact compound produced will depend upon the reaction stoichiometryand the starting materials with a skilled addressee being able to adjusteither of these variables to produce the desired final product.

In addition it is desired that the linker X be extended to besignificantly longer than the compounds readily accessible by the routedetailed above it is possible to elaborate the carboxy group (such as bystandard peptide chemistry techniques) to introduce further amino acidgroups to the chain. The methods of achieving reactions of this type arewell within the skill of a skilled addressee in the area.

Examples of compounds of formula (I) that may be produced using themethodology described above include:

or a metal complex thereof.

These compounds may then be further elaborated to produce compounds offormula (II) which contain a molecular recognition moiety by reaction ofthe carboxyl terminus (or an activated form thereof) with a suitablereactive element on a molecular recognition moiety under suitablecoupling conditions. An example of such a reaction would be one in whicha coupling between the carboxyl portion of the compound formula (I) isconducted with the N termini of a peptide or protein (such as theN-terminus of a biologically active peptide) to form a peptide linkagesuch that the metal ligand becomes bound (via the linker) to themolecular recognition unit.

Alternatively the molecules of formula (I) may be reacted with moleculesto introduce a ‘spacer moiety” prior to addition of the molecularrecognition portion of the molecular recognition moiety. Thus forexample the compounds of formula (I) may be functionalised, for examplethrough the carboxyl moiety to form one or more peptidic linkages toproduce as a synthetic intermediate a compound of formula (I) with aspacer moiety attached (the spacer moiety forming part of the molecularrecognition moiety in the final compound). Examples of compounds of thistype include:

As can be seen in each of these compounds the compound of formula (I)has been elaborated by addition of a ‘spacer moiety”. In the firstcompound a tyrosine moiety has been added to one side of the molecule.In the second compound the peptide Tyr-aHX-Cys-NH₂ has been added to oneside of the molecule and in the third compound the peptideTyr-aHX-Cys-NH₂ has been added to both sides of the molecule.

As stated above the compound of formula (I) or intermediate compounds asdiscussed above may then be further elaborated by addition of amolecular recognition moiety (or molecular recognition portion) as thecase may be to form compounds of formula (II).

Examples of compounds of formula (II) include:

In principle any of a wide range of biologically active molecularrecognition units may be employed in the present application with theonly limitation being that the molecular recognition moiety used mustcontain a functionality capable of being bound (either directly orthrough a spacer) to the compound of formula (I). Whilst a number ofdifferent functional groups may be contemplated (such as the maleimidopropionate group on RGDfK above which couples with a thiol moiety) themolecular recognition moiety preferably has an N terminus for couplingthrough the carboxyl residue of the compound of the invention asdiscussed above. The coupling reactions may be carried out in ways wellknown in the art and employ peptide synthesis techniques well known inthe art which may involve either solid phase or liquid phase peptidesynthesis techniques to be used. In some instances the nitrogen atoms ofthe ligand may be protected prior to peptide coupling using standardnitrogen protecting groups in order to facilitate smooth coupling. Ifthis is done any suitable nitrogen protecting group may be used with theN-tert-butoxy carbonyl group (t-boc) being found to be particularlyuseful. Upon completion the protective groups may be removed usingtechniques well known in the art.

The formation of the metal complexes of the compounds synthesised inthis way is carried out using techniques well known in the art.

As discussed above the compounds of the invention are useful as theyeither have, or can be modified to contain a molecular recognitionmoiety. The compounds of formula (II) containing a radionuclidecomplexed with the ligand may be used in either radiotherapy or indiagnostic imaging applications. In each instance both therapy anddiagnostic imaging will rely on the molecular recognition moiety beinginvolved in facilitating the localisation of the complex containing theradionuclide in the desired tissues or organs of the subject beingtreated/imaged.

Thus for example in relation to the use of the radiolabelled compoundsof formula (II) it is anticipated that these will be used byadministration of an effective amount of the radiolabelled compound to asubject followed by monitoring of the subject after a suitable timeperiod to determine if the radiolabelled compound has localised at aparticular location in the body or whether the compound is broadlyspeaking evenly distributed through the body. As a general rule wherethe radio labelled compound is localised in tissue or an organ of thebody this is indicative of the presence in that tissue or organ ofsomething that is recognised by the particular molecular recognitionmoiety used.

Accordingly judicious selection of a molecular recognition moiety ormolecular recognition portion is important in determining the efficacyof any of the radiolabelled compounds of the invention in diagnosticimaging applications. In this regard a wide range of molecularrecognition moieties or molecular recognition portions are known in theart which are well characterised and which are known to selectivelytarget certain receptors in the body. In particular a number ofmolecular recognition moieties or molecular recognition portions areknown that target tissue or organs when the patient is suffering fromcertain medical conditions. Examples of molecular recognition moietiesor molecular recognition portions that are known and may be used in thisinvention include Octreotate, octreotide, [Tyr³]-octreotate,[Tyr¹]-octreotate, bombesin, bombesin(7-14), gastrin releasing peptide,single amino acids, penetratin, annexin V, TAT, cyclic RGD, glucose,glucosamine (and extended carbohydrates), folic acid, neurotensin,neuropeptide Y, cholecystokinin (CCK) analogues, vasoactive intestinalpeptide (VIP), substance P, alpha-melanocyte-stimulating hormone (MSH).For example, certain cancers are known to over express somatostatinreceptors and so the molecular recognition moiety may be one whichtargets these receptors. An example of a molecular recognition moietiesor molecular recognition portions of this type is [Tyr³]-octreotate.Another example of a molecular recognition moieties or molecularrecognition portions is cyclic RGD which is an integrin targeting cyclicpeptide. In other examples a suitable molecular recognition moieties ormolecular recognition portions is bombesin which is known to targetbreast and pancreatic cancers.

The monitoring of the subject for the location of the radiolabelledmaterial will typically provide the analyst with information regardingthe location of the radiolabelled material and hence the location of anymaterial that is targeted by the molecular recognition moiety (such ascancerous tissue). An effective amount of the compounds of the inventionwill depend upon a number of factors and will of necessity involve abalance between the amount of radioactivity required to achieve thedesired radio imaging effect and the general interest in not exposingthe subject (or their tissues or organs) to any unnecessary levels ofradiation which may be harmful.

The methods of treatment of the present invention involve administrationof a compound of formula (II) complexed to a radionuclide. The compoundsof formula (II) contain a molecular recognition moiety in order todeliver the radionuclide to the desired location in the body where itsmode of action is desired. As discussed above examples of such molecularrecognition moieties are known in the art and a skilled artisan canselect the appropriate molecular recognition moiety to target thedesired tissue in the body to be treated.

A therapeutically effective amount can be readily determined by anattending clinician by the use of conventional techniques and byobserving results obtained under analogous circumstances. In determiningthe therapeutically effective amount a number of factors are to beconsidered including but not limited to, the species of animal, itssize, age and general health, the specific condition involved, theseverity of the condition, the response of the patient to treatment, theparticular radio labelled compound administered, the mode ofadministration, the bioavailability of the preparation administered, thedose regime selected, the use of other medications and other relevantcircumstances.

In addition the treatment regime will typically involve a number ofcycles of radiation treatment with the cycles being continued until suchtime as the condition has been ameliorated. Once again the optimalnumber of cycles and the spacing between each treatment cycle willdepend upon a number of factors such as the severity of the conditionbeing treated, the health (or lack thereof) of the subject being treatedand their reaction to radiotherapy. In general the optimal dosage amountand the optimal treatment regime can be readily determined by a skilledaddressee in the art using well known techniques.

In using the compounds of the invention they can be administered in anyform or mode which makes the compound available for the desiredapplication (imaging or radio therapy). One skilled in the art ofpreparing formulations of this type can readily select the proper formand mode of administration depending upon the particular characteristicsof the compound selected, the condition to be treated, the stage of thecondition to be treated and other relevant circumstances. We refer thereader to Remingtons Pharmaceutical Sciences, 19th edition, MackPublishing Co. (1995) for further information.

The compounds of the present invention can be administered alone or inthe form of a pharmaceutical composition in combination with apharmaceutically acceptable carrier, diluent or excipient. The compoundsof the invention, while effective themselves, are typically formulatedand administered in the form of their pharmaceutically acceptable saltsas these forms are typically more stable, more easily crystallised andhave increased solubility.

The compounds are, however, typically used in the form of pharmaceuticalcompositions which are formulated depending on the desired mode ofadministration. The compositions are prepared in manners well known inthe art.

The invention in other embodiments provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. In sucha pack or kit can be found at least one container having a unit dosageof the agent(s). Conveniently, in the kits, single dosages can beprovided in sterile vials so that the clinician can employ the vialsdirectly, where the vials will have the desired amount and concentrationof compound and radio nucleotide which may be admixed prior to use.Associated with such container(s) can be various written materials suchas instructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, imaging agents or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

The compounds of the invention may be used or administered incombination with one or more additional drug(s) that are anti-cancerdrugs and/or procedures (e.g. surgery, radiotherapy) for the treatmentof the disorder/diseases mentioned. The components can be administeredin the same formulation or in separate formulations. If administered inseparate formulations the compounds of the invention may be administeredsequentially or simultaneously with the other drug(s).

In addition to being able to be administered in combination with one ormore additional drugs that include anti-cancer drugs, the compounds ofthe invention may be used in a combination therapy. When this is donethe compounds are typically administered in combination with each other.Thus one or more of the compounds of the invention may be administeredeither simultaneously (as a combined preparation) or sequentially inorder to achieve a desired effect. This is especially desirable wherethe therapeutic profile of each compound is different such that thecombined effect of the two drugs provides an improved therapeuticresult.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of micro-organisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents that delay absorptionsuch as aluminium monostearate and gelatin.

If desired, and for more effective distribution, the compounds can beincorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes, and microspheres.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

If desired, and for more effective distribution, the compounds can beincorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes, and microspheres.

The active compounds can also be in microencapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminiummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

As discussed above, the compounds of the embodiments may be useful fortreating and/or detecting proliferative diseases. Examples of such cellproliferative diseases or conditions include cancer (include anymetastases), psoriasis, and smooth muscle cell proliferative disorderssuch as restenosis. The compounds of the present invention may beparticularly useful for treating and/or detecting tumours such as breastcancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, headand/or neck cancer, or renal, gastric, pancreatic cancer and braincancer as well as hematologic malignancies such as lymphoma andleukaemia. In addition, the compounds of the present invention may beuseful for treating and/or detecting a proliferative disease that isrefractory to the treatment and/or detecting with other anti-cancerdrugs; and for treating and/or detecting hyperproliferative conditionssuch as leukaemia's, psoriasis and restenosis. In other embodiments,compounds of this invention can be used to treat and/or detectpre-cancer conditions or hyperplasia including familial adenomatouspolyposis, colonic adenomatous polyps, myeloid dysplasia, endometrialdysplasia, endometrial hyperplasia with atypia, cervical dysplasia,vaginal intraepithelial neoplasia, benign prostatic hyperplasia,papillomas of the larynx, actinic and solar keratosis, seborrheickeratosis and keratoacanthoma.

Synthesis of Compounds of the Invention

The agents of the various embodiments may be prepared using the reactionroutes and synthesis schemes as described below, employing thetechniques available in the art using starting materials that arereadily available. The preparation of particular compounds of theembodiments is described in detail in the following examples, but theartisan will recognize that the chemical reactions described may bereadily adapted to prepare a number of other agents of the variousembodiments. For example, the synthesis of non-exemplified compounds maybe successfully performed by modifications apparent to those skilled inthe art, e.g. by appropriately protecting interfering groups, bychanging to other suitable reagents known in the art, or by makingroutine modifications of reaction conditions. A list of suitableprotecting groups in organic synthesis can be found in T. W. Greene'sProtective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, 1991. Alternatively, other reactions disclosed herein or known inthe art will be recognized as having applicability for preparing othercompounds of the various embodiments.

Reagents useful for synthesizing compounds may be obtained or preparedaccording to techniques known in the art.

General Synthetic Scheme

Scheme 1 is a general synthetic scheme outlining the procedures for themanufacture of compounds of the invention of general formula (I). Thisgeneral procedure can be modified to produce other compounds of theinvention with different linking moieties X of the cyclic dicarbonyl(III) by appropriate modification of the starting materials and reagentsused. A skilled addressee would readily be able to make these changes.

As can be seen in scheme 1 an appropriately nitrogen containingmacrocyclic ligand (V) (which may or may not be complexed with a metal)is reacted with a suitable cyclic dicarbonyl (III) containing a desiredlinking moiety X under basic conditions to provide the dicarbonyl metalchelating agent (I). The dicarbonyl metal chelating agent (I) has afunctional group Y for further conjugation with a suitable molecularrecognition moiety.

This scheme outlines a synthesis wherein Z is an oxygen or sulfur atomor a group of formula NR². When Z is an oxygen atom, a terminalcarboxylic acid handle is formed for further elaboration. Alternatively,when Z is a sulfur atom, a terminal carboxylic thiol is formed forfurther elaboration by methods well known in the art. When Z is NR² theterminal amide is formed. The scheme may be modified to producecompounds within the scope of the present disclosure in which the cyclicdicarbonyl (III) may also act as a base in the reaction shown above.Other variations to arrive at the desired final product is within theskill of a skilled addressee in the art.

Scheme 2 demonstrates an alternative synthesis of the compounds of theinvention. As shown in scheme 2 an alternative synthesis involves theprovision of a suitably activated linear dicarbonyl compound in whichone of the carbonyl moieties has been activated by providing thecarbonyl with a suitable leaving group. This is reacted with eth aminecompound under suitable reaction conditions leading to displacement ofthe leaving group and formation of the desired final product.

The nitrogen containing macrocyclic metal chelating ligand L for use inthe syntheses described above can be any of a range of macrocyclic metalligands known in the art as discussed above. In the schemes above themetal ligand can be reacted with the anhydride in free form or be boundto a metal ion thereby deactivating the nucleophilicity of theheteroatoms in the macrocycle prior to reaction. One example of such ametal chelator is 1,8-(NH₂)₂sar. 1,8-(NH₂)₂sar can coordinate with ametal ion to substantially deactivate the six secondary amines in themacrocycle and thereby selectively allowing the two “free” primaryamines to react.

The compounds of formula (II) may be produced from the compounds offormula one using standard peptide coupling techniques in which thecarboxy termini of the compounds of formula (I) is reacted with theamine terminus of a molecular recognition moiety under standardconditions to produce the coupled product.

Synthetic procedures for the synthesis of selected compounds of formula(I) are detailed below.

EXAMPLES

In the examples described below, unless otherwise indicated, alltemperatures in the following description are in degrees Celsius and allparts and percentages are by weight, unless indicated otherwise.

Various starting materials and other reagents were purchased fromcommercial suppliers, such as Aldrich Chemical Company or LancasterSynthesis Ltd., and used without further purification, unless otherwiseindicated. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) werepurchased from Aldrich in SureSeal bottles and used as received. Allsolvents were purified by using standard methods in the art, unlessotherwise indicated. SP Sephadex C25 and DOWEX 50 w×2 200-400 meshcation exchange resin was purchased from Aldrich. Fmoc-L-amino acids,HATU, HCTU and 2-chlorotrityl resin were purchased from GL Biochem Ltd(Shanghai, China). Fmoc-Lys(iv-Dde)-OH and Fmoc-D-amino acids werepurchased from Bachem AG (Switzerland). Fmoc-Pal-PEG-PS resin waspurchased from Applied Biosystems (Foster City, Calif.). Nova PEG RinkAmide resin was purchased from NovaBiochem, Darmstadt, Germany.[Co((NO₂)₂sar)]Cl₃, [Co((NH₂)₂sar)]Cl₃, (NH₂)₂sar,[Cu(NH₃)₂sar](CF₃SO₃)₄ were prepared according to establishedprocedures. (1) Geue, R. J.; Hambley, T. W.; Harrowfield, J. M.;Sargeson, A. M.; Snow, M. R. J. Am. Chem. Soc. 1984, 106, 5478-5488. (2)Bottomley, G. A.; Clark, I. J.; Creaser, I. I.; Engelhardt, L. M.; Geue,R. J.; Hagen, K. S.; Harrowfield, J. M.; Lawrance, G. A.; Lay, P. A.;Sargeson, A. M.; See, A. J.; Skelton, B. W.; White, A. H.; Wilner, F. R.Aust. J. Chem. 1994, 47, 143-179 and (3) Bernhardt, P. V.; Bramley, R.;Engelhardt, L. M.; Harrowfield, J. M.; Hockless, D. C. R.;Korybut-Daszkiewicz, B. R.; Krausz, E. R.; Morgan, T.; Sargeson, A. M.;Skelton, B. W.; White, A. H. Inorg. Chem. 1995, 34, 3589-3599.

The reactions set forth below were performed under a positive pressureof nitrogen, argon or with a drying tube, at ambient temperature (unlessotherwise stated), in anhydrous solvents, and the reaction flasks arefitted with rubber septa for the introduction of substrates and reagentsvia syringe. Glassware was oven-dried and/or heat-dried.

Work-ups were typically done by doubling the reaction volume with thereaction solvent or extraction solvent and then washing with theindicated aqueous solutions using 25% by volume of the extraction volume(unless otherwise indicated). Product solutions were dried overanhydrous sodium sulfate prior to filtration, and evaporation of thesolvents was under reduced pressure on a rotary evaporator and noted assolvents removed in vacuo. Flash column chromatography [Still et al, J.Org. Chem., 43, 2923 (1978)] was conducted using E Merck-grade flashsilica gel (47-61 mm) and a silica gel:crude material ratio of about20:1 to 50:1, unless otherwise stated. Hydrogenolysis was done at thepressure indicated or at ambient pressure.

Mass spectra were recorded in the positive ion mode on an Agilent 6510Q-TOF LC/MS Mass Spectrometer coupled to an Agilent 1100 LC system(Agilent, Palo Alto, Calif.). Data were acquired and reference masscorrected via a dual-spray electrospray ionisation source, using thefactory-defined calibration procedure. Each scan or data point on theTotal Ion Chromatogram is an average of 9652 transients, producing 1.02scans s⁻¹. Spectra were created by averaging the scans across each peak.Mass spectrometer conditions: fragmentor: 200-300 V; drying gas flow: 7L/min; nebuliser: 30 psi; drying gas temp: 325° C.; V_(cap): 4000 V;skimmer: 65 V; OCT R_(f)V: 750 V; scan range acquired: 150-3000 m/z.

HPLC-MS traces were recorded using an Agilent Eclipse Plus C18 column (5μm, 2.1×150 mm) coupled to the Agilent 6510 Q-TOF LC/MS MassSpectrometer described above. 1 μL aliquots of each sample were injectedonto the column using the Agilent 1100 LC system, with a flow rate of0.5 mL/min. Data acquisition parameters are the same as those describedabove for mass spectra, with the exception of the fragmentor (fragmentorvoltage: 100 V).

NMR spectra were recorded on a Varian FT-NMR 500 spectrometer operatingat 500 MHz for ¹H NMR and 125.7 MHz for ¹³C-NMR. NMR spectra areobtained as D₂O solutions (reported in ppm), using acetone as thereference standard (2.22 ppm and 30.89 ppm respectively). Other NMRsolvents were used as needed. When peak multiplicities are reported, thefollowing abbreviations are used: s=singlet, d=doublet, t=triplet,m=multiplet, br=broadened, dd=doublet of doublets, dt=doublet oftriplets. Coupling constants, when given, are reported in Hertz.

Semi-preparative HPLC purifications were performed using an Agilent 1200Series HPLC system with a 5 mL/min flow rate. Solvent gradients andcolumn specifications are described in the examples. An automatedAgilent 1200 fraction collector collected 1-3 mL fractions and fractioncollection was based on UV-Vis detection at 214 or 220 nm, with a lowerthreshold limit between 100-400 mAU. Each fraction was analysed using MSand analytical HPLC.

Analytical HPLC traces were acquired using an Agilent 1200 Series HPLCsystem and an Agilent Zorbax Eclipse XDB-C₁₈ column (4.6×150 mm, 5 μm)with a 1 mL/min flow rate and UV spectroscopic detection at 214 nm, 220nm and 270 nm.

UV-Vis spectra were acquired on a Cary 300 Bio UV-Vis spectrophotometer,from 800-200 nm at 0.500 nm data intervals with a 300.00 nm/min scanrate.

Voltametric experiments were performed with an Autolab (Eco Chemie,Utrecht, Netherlands) computer-controlled electrochemical workstation. Astandard three-electrode arrangement was used with a glassy carbon disk(d, 3 mm) as working electrode, a Pt wire as auxiliary electrode and aAg/AgCl reference electrode (silver wire in H₂O (KCl (0.1 M) AgNO₃ (0.01M)). Scan rate: 100 mV/s, sample interval: 1.06 mV, sensitivity: 1×10⁻⁴A.

HPLC traces of radiolabelled peptides were acquired using a WatersComosil C18 column (4.6×150 mm) coupled to a Shimadzu LC-20AT with asodium iodide scintillation detector and a UV-Vis detector. 100 μLaliquots of each radiolabelled sample were injected onto the column,using a flow rate of 1 mL/min.

The following examples are intended to illustrate the embodimentsdisclosed and are not to be construed as being limitations thereto.Additional compounds, other than those described below, may be preparedusing the following described reaction scheme or appropriate variationsor modifications thereof.

Example 1 [Cu((1-NH₃)(8-NHCO(CH₂)₃COOH)Sar)](NO₃)₃ (Also Called[CuL¹](NO₃)₃) and [Cu(1,8-NHCO(CH₂)₃COOH)₂Sar](CF₃SO₃)Cl (Also Called[CuL²](CF₃SO₃)Cl)

A solution of [Cu(NH₃)₂Sar](CF₃SO₃)₄ (1.5 g, 1.53 mmol) in anhydrousN,N-dimethylacetamide (12 mL) was heated under an atmosphere of nitrogento 70° C. Glutaric anhydride (0.19 g, 1.64 mmol) anddiisopropylethylamine (600 μL) were added and the solution was heated at70° C. for two hours. The solution was cooled and water (20 mL) wasadded. The solution was applied to a column of SP Sephadex C-25 cationexchange (Nat form, 30×5 cm). The column was eluted with 0.05 M sodiumcitrate solution to separate three components. (Chromatographic yield:Fraction 1˜40%, fraction 2˜40%, fraction 3˜20%.) Each fraction wasapplied separately to a DOWEX 50 W×2 cation exchange column (H⁺ form,10×5 cm). The column was washed with water (500 mL) and 1 M HCl solution(500 mL) and then eluted with 4 M HCl (350 mL) and the eluent wasevaporated to dryness under reduced pressure at 40° C. Fraction 1:[Cu(1,8-NHCO(CH₂)₃COOH)₂Sar]Cl₂.xHCl (1.00 g) MS: [CuC₂₄H₄₅N₈O₆]⁺m/z=604.2732 (experimental), 604.2764 (calculated). Fraction 2:[Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar]Cl₃.xHCl (0.82 g) MS: [CuC₁₉H₃₉N₈O₃]⁺m/z=490.2439 (experimental), 490.2447 (calculated). The dark blueresidue from fraction 2 was dissolved in distilled water (30 mL).Concentrated nitric acid (2 mL) was added and the solution wasconcentrated by rotary evaporation until crystallisation commenced. Themixture was cooled at 5° C. for 30 mins before the light blue crystalswere collected by filtration. [Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar](NO₃)₃:0.13 g, 13% isolated yield.

Crystals suitable for X-ray diffraction were grown from evaporation of asolution of [Cu(1-NH₃)(8-NHCO(CH₂)₃COOH)Sar]Cl₃.xHCl (20 mg) in ˜1 MHNO₃ (2 mL) at ambient temperature.

Crystals of [Cu(1,8-NHCO(CH₂)₃COOH)₂Sar](CF₃SO₃)Cl were suitable forX-ray diffraction studies and were formed as follows:[Cu(1,8-NHCO(CH₂)₃COOH)₂Sar]Cl₂.xHCl (0.45 g) was dissolved in water (2mL) and a solution of silver triflate (0.33 g in 2 mL water) was added.This solution was filtered twice (MilliQ syringe filter (0.45 μm)) andevaporated to dryness under reduced pressure to give a dark blue-purpleresidue. The residue was redissolved in water (8 mL) and over the courseof 10 min, blue crystals precipitated from this solution. These werecollected and dried by filtration.Cu(1,8-NHCO(CH₂)₃COOH)₂Sar](CF₃SO₃)Cl: 0.13 g. Crystals suitable forX-ray diffraction were grown from evaporation of a solution ofCu(1,8-NHCO(CH₂)₃COOH)₂Sar](CF₃SO₃)Cl (30 mg) in water (6 mL) at ambienttemperature. Crystal data: [CuL¹](NO₃)₃ C₂₅H₄₁N₁₁O₁₂Cu, M=679.17,T=130.0 (2) K, λ□=0.71069, monoclinic, space group P2_(1/C) a=8.345 (5)b=12.231 (5), c=26.941 (5) Å, b=93.658 (5) °, V=2744 (2) Å³, Z=4,D_(c)=1.644 mg M⁻³μ(Mo-Kα) 0.879 mm⁻¹, F(000)=1428, crystal size0.35×0.3×0.01 mm. 17656 reflections measured, 6107 independentreflections (R_(int)=0.17), the final R was 0.067 [I>2□(I)] and wR(F²)was 0.1524. [CuL²](CF₃SO₃)Cl C₁₉H₄₆ClCuF₃N₈O₉S, M=790.75, T=130.0 (2) K,λ□=0.71069, orthorhombic, space group C222₁ a=12.4608 (13) b=20.445 (2),c=13.2263 (14) Å, V=3369.6 (6) Å³, Z=4, D_(c)=1.559 mg M⁻³ μ(Mo-Kα)0.879 mm⁻¹, F(000)=1652, crystal size 0.40×0.30×0.20 mm. 8848reflections measured, 2973 independent reflections (R_(int)=0.0334), thefinal R was 0.0542 [I>2σ(I)] and wR(F²) was 0.1471. Structures obtainedand solved by Assoc. Prof. Jonathan M. White.

Microanalysis: [CuL¹](NO₃)₃: CuC₁₉H₄₁N₁₁O₁₂—C 34.86%, H 6.40%, N 24.76%(experimental); —C 33.60%, H 6.08%, N 22.69% (calculated);[CuL²](CF₃SO₃)Cl: CuC₂₅H₄₆N₈O₉SClF₃—C 38.06%, H 5.92%, N 14.20%, S 3.88%(experimental); —C 37.97%, H 5.86%, N 14.17%, S 4.06% (calculated).

UV-vis: [Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar](NO₃)₃ in water, pH 4,λ_(max)=658 nm, ε=140 M⁻¹ cm⁻¹; [Cu(1,8-NHCO(CH₂)₃COOH)₂Sar](CF₃SO₃)Clin water, pH=4, λ_(max)=655 nm, ε=146 M⁻¹ cm⁻¹

Electrochemistry: [Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar](NO₃)₃ (1 mM) in anaqueous solution of NaBF₄ (100 mM), pH 3.5, E_(red)=−1.086 (vs[Fe(CN)₆]^(3-/4-), E°=0).

Example 2 (1-NH₃Cl)(8-NHCO(CH₂)₃COOH)Sar: (Also Called L¹.HCl)

A solution of [Cu(1-NH₃)(8-NHCO(CH₂)₃COOH)Sar]Cl₃.xHCl (0.44 g, ˜0.73mmol based on x=0) in water (4 mL) was deoxygenated by purging with N₂gas for 20 mins. Sodium sulfide (0.6 g) was added and the solution wasstirred overnight at room temperature (under an atmosphere of nitrogengas). After addition of sodium sulfide, the solution turned a darkgreen. After ˜16 hours, a black-brown precipitate was present and thesolution appeared a light yellow-green. This mixture was filtered(Whatman Filter Paper 1) and the filtrate diluted with 1 M HCl (250 mL)resulting in the formation of a cloudy, white precipitate. The mixturewas filtered (MilliQ syringe filters (0.45 m)) and applied to a DOWEX 50W×2 cation exchange column (H⁺ form, 10×5 cm). The column was washedwith 1 M HCl solution (750 mL) (to remove Na₂S) and then eluted with 4 MHCl solution (400 mL). The eluent was evaporated to dryness underreduced pressure to give a clear residue with a slight blue tinge.Because this residue was still slightly blue, the above process wasrepeated. The final solution was evaporated to dryness to give a clear,colourless residue. (1-NH₃Cl(8-NHCO(CH₂)₃COOH)Sar.xHCl: 0.30 g, 89% MS:[C₁₉H₄₀N₈O₃]⁺429.3372 (experimental), 429.3296 (calculated). ¹H NMR: δ1.852, m, 2H, βCH₂ (with respect to COOH); 2.358, t, ³J=7.54, 2H,glutarate CH₂; 2.417, t, ³J=7.18, 2H glutarate CH₂; 3.181, broad s, 6H,cage CH₂; 3.315, broad s, 12H, cage CH₂; 3.694, broad s, 6H, cage CH₂.¹³C NMR: δ 20.36, 33.46, 35.54 (glutarate CH₂); 46.46, 48.39, 50.66,51.18, 55.23, 56.54 (cage); 177.937, 178.516 (CO).

Example 3 (8-NHCO(CH₂)₃COOH)(x-NCO₂-t-Bu)_(4.5)Sar: (Also CalledL¹-(t-BOC)₃₋₅)

(1-NH₃Cl)(8-NHCO(CH₂)₃COOH)Sar.xHCl was converted to atrifluoromethanesulfonate salt to increase its solubility inN,N-dimethylacetamide. In brief, (1-NH₄Cl)(8-NHCO(CH₂)₃COOH)Sar.xHCl(0.20 g, 0.43 mmol) was dissolved in water (5 mL) and silver triflate(0.11 g, 0.43 mmol) was added, precipitating silver chloride. Thesolution was filtered (MilliQ 0.45 μm syringe filter) and evaporated todryness under reduced pressure to give a colourless, clear hydroscopicresidue. (1-NH₄CF₃SO₃)(8-NHCO(CH₂)₃COOH)Sar.xH₂O: 0.31 g.

(1-NH₃CF₃SO₃)(8-NHCO(CH₂)₃COOH)Sar.xH₂O (80 mg, 0.138 mmol based on x=0)was dissolved in a solution of N,N-dimethylacetamide:water (4:1) (10mL). Di-tert-butyldicarbonate (0.25 g, 1.15 mmol) anddiisopropylethylamine (100 μL) were added and the solution was stirredunder an atmosphere of nitrogen gas for 30 min. After this time, thesolvent was removed under high vacuum at ˜40° C. The residue wasdissolved in acetonitrile (15 mL), filtered (MilliQ 0.45 μm syringefilter) and lyophilised to remove traces of N,N-dimethylacetamide. Oncethe crude compound was dry, it was dissolved in a solution of A:B(70:30) (A=milliQ water containing 0.1% trifluoroacetic acid,B=acetonitrile containing 0.1% trifluoroacetic acid) (5 mL), filtered(MilliQ 0.45 m syringe filter), and applied to a C18 cartridge (AlltechMaxi-Clean C18 900 mg). The cartridge was washed sequentially with 5 mLA, 5 mL 10% B in A and 5 mL 20% B in A. It was then eluted with 5 mL 80%B in A, and 1 mL fractions were collected. Most of the desired compound(>95%) was collected in the first two fractions. These were lyophilisedto yield a mixture of t-BOC protected isomers, and the degree ofprotection ranged from 3-5 t-BOC groups per molecule of cage compound.(8-NHCO(CH₂)₃COOH)(x-NCOO-t-Bu)_(4.5)Sar: 30 mg, 25% yield. MS:[C₁₉H₄₁N₈O₃(C₅H₈O₂)₃]⁺729.4879 (experimental), 729.4869 (calculated);[C₁₉H₄₁N₈O₃(C₅H₈O₂)₄]⁺829.5414 (experimental), 829.5414 (calculated);[C₁₉H₄₁N₈O₃(C₅H₈O₂)₅]⁺929.5942 (experimental), 929.5918 (calculated).

Example 4 (1,8-NHCO(CH₂)₃COOH)₂Sar: (Also Called L²)

A solution of [Cu(1,8-NHCO(CH₂)₃COOH)₂Sar]Cl₂.xHCl (1.0 g, ˜1.48 mmolbased on x=0) in water (20 mL) was deoxygenated by purging with N₂ gasfor 20 mins. Sodium sulfide (1.3 g) was added and the solution wasstirred overnight at room temperature (under an atmosphere of nitrogengas). After ˜16 hours, the solution was green with a black-brownprecipitate. Another portion of sodium sulfide (1.4 g) was added and thesolution stirred overnight at room temperature. After this time, thesolution appeared light yellow. This mixture was filtered (WhatmanFilter Paper 1) and the filtrate diluted with 1 M HCl (250 mL) resultingin the formation of a cloudy, white precipitate. The mixture wasfiltered (MilliQ syringe filters (0.45 μm)) and applied to a DOWEX 50W×2 cation exchange column (H⁺ form, 10×5 cm). The column was washedwith 1 M HCl solution (750 mL) (to remove Na₂S) and then eluted with 4 MHCl solution (400 mL). The eluent was evaporated to dryness underreduced pressure to give a clear, colourless residue.(1,8-NHCO(CH₂)₃COOH)₂Sar.xHCl: 0.255 g, 32% MS: [C₂₄H₄₇N₈O₆]⁺543.3686(experimental), 534.3619 (calculated). ¹H NMR: δ 1.856, m, 4H, βCH₂(with respect to COOH); 2.336, t, ³J=7.47, 4H, glutarate CH₂; 2.411, t,³J=7.19, 4H glutarate CH₂; 3.210, broad s, 12H, cage CH₂; 3.439, broads, 12H, cage CH₂; ¹³C NMR: δ 20.65, 33.43, 35.46 (glutarate CH₂); 47.40,51.44. 56.23 (cage); 177.80, 178.44 (CO).

Example 5 L²-(t-Boc)₄

L².xHCl (0.17 g, 0.31 mmol based on x=0) was dissolved in water (3 mL).Di-tert-butyldicarbonate (1 g, ˜20 equivalents) in acetonitrile (7 mL)and triethylamine (0.5 mL) were added. The acetonitrile phase was notmiscible with the aqueous phase, but the reaction was stirred vigorouslyunder an inert atmosphere of nitrogen gas for 2 hours. After this time,the solvent was removed under reduced pressure and the remaining residuewas dried under high vacuum at 45° C. for 2 hours. The dried residue wasredissolved in a solution of A:B (90:10), filtered (MilliQ 0.45 μmsyringe filter), and applied to a C18 cartridge (Alltech Maxi-Clean C18900 mg). The cartridge was washed sequentially with 5 mL A and 5 mL 20%B in A. It was then eluted with 5 mL 50% B in A. This fraction waslyophilised to yield mainly L²-(t-Boc)₄. L¹-(t-Boc)₃₋₅: 30 mg, 10%yield. MS: [C₄₄H₇₉N₈O₁₄]⁺943.68 (experimental), 943.57 (calculated);

Example 6 Sar-octreotate (L¹-Tyr³-Octreotate)

Linear [Tyr³]-octreotate (linear OCT) peptide(dPhe-Cys-Tyr-dTrp-Lys-Thr-Cys-Thr-OH (Seq ID No:1) was synthesised on2-chlorotrityl chloride resin using standard Fmoc solid phase peptidesynthesis procedures. An excess of resin (0.06 g, ˜0.8 mmol/g) wasswelled in N,N-dimethylformamide (DMF). L¹-(t-Boc)₃₋₅ (25 mg, 0.03mmol), HCTU (20 mg, 0.05 mmol) and diisopropylethylamine (20 μL) in DMF(1 mL) were added to the resin and the mixture was stirred and left toreact overnight. The reaction supernatant was then drained and the resinwashed with DMF (3×5 mL) and dichloromethane (DCM) (3×5 mL). The resinwas transferred to a falcon tube and trifluoroacetic acid (5 mL),deionised water (0.15 mL) and triisopropylsilane (0.15 mL) were added.The falcon tube was placed on a shaker for 40 min. The peptide materialwas precipitated from the solution using diethyl ether (15 mL) and themixture was centrifuged (3 mins, 3000 rpm). The supernatant wasdiscarded and the precipitate dissolved in A:B (70:30). This solutionwas filtered (MilliQ 0.45 μm syringe filter) and lyophilised.

The crude peptide material was purified by semi-preparative reversephase HPLC (Eclipse XDB-C18 5 μm 9.5×250 mm column) using a linear 1%A→B/min gradient. Linear L¹-Tyr³-octreotate eluted at 30 min (determinedby ESI-MS) and fractions containing linear L¹-Tyra-octreotate werelyophilised. The dried fractions were then redissolved in ammoniumacetate (25 mM, pH 6.5, 8 mL) and an excess of 2,2-dithiodipyridine (12mg) was added. The solution was then applied to a semi-preparativereverse phase HPLC column, and purified using a linear 1% A→B/mingradient. Cyclic L¹-Tyr³-octreotate eluted at 30 min and fractionscontaining cyclic L¹-Tyr³-octreotate were lyophilised.L¹-Tyr³-octreotate: 1-2 mg; HPLC retention time: 12.99 min (lineargradient, 0→60% B in A over 25 min); MS: [C₆₈H₁₀₅N₁₈O₁₄S₂]³⁺ 487.25(experimental), 487.25 (calculated); [C₆₈H₁₀₄N₁₈O₁₄S₂]²⁺730.37(calculated), 730.37 (experimental).

Example 7 CuSar-octreotate (Cu L¹-Tyr³-Octreotate)

[Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar]Cl₃.xHCl was converted to atrifluoromethanesulfonate salt to increase its solubility inN,N-dimethylacetamide. In brief,[Cu(1-NH₃)(8-NHCO(CH₂)₃COOH)Sar]Cl₃.xHCl (0.14 g, 0.23 mmol) wasdissolved in water (5 mL) and silver triflate (0.18 g, 0.70 mmol) wasadded, precipitating silver chloride. The solution was filtered (MilliQ0.45 μm syringe filter) and evaporated to dryness under reduced pressureto give a sticky, blue hydroscopic residue.[Cu(1-NH₃)(8-NHCO(CH₂)₃COOH)Sar](CF₃SO₃)₃.xH₂O: 0.21 g.

Linear octreotate peptide on resin (0.10 g, ˜0.8 mmol/g) was swelled indimethylformamide. Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar](CF₃SO₃)₃ (30 mg, 0.03mmol), HCTU (40 mg, 0.1 mmol) and diisopropylethylamine (40 μL) in DMF(1 mL) were added to the resin and the mixture was stirred and left toreact for 3 hr. The reaction supernatant was then drained and the resinwashed with dimethylformamide (3 x 5 mL). A second coupling wasperformed. Cu(1-NH₄)(8-NHCO(CH₂)₃COOH)Sar](CF₃SO₃)₃ (10 mg, 0.01 mmol),HCTU (20 mg, 0.05 mmol) and diisopropylethylamine (20 μL) in DMF (1 mL)were added to the resin and the mixture was stirred and left to reactfor 40 min. The reaction supernatant was then drained and the resinwashed with dimethylformamide (3×5 mL) and dichloromethane (3×5 mL). Theresin was transferred to a falcon tube and trifluoroacetic acid (5 mL),distilled water (0.15 mL) and triisopropylsilane (0.15 mL) were added.The falcon tube was placed on a shaker for 40 min. The peptide materialwas precipitated from the solution using diethyl ether (15 mL) and themixture was centrifuged (3 min, 3000 rpm). The supernatant was discardedand the precipitate dissolved in A:B (70:30). This solution was filtered(MilliQ 0.45 μm syringe filter) and lyophilised to give pale bluematerial.

The crude peptide material was purified by semi-preparative reversephase HPLC (Eclipse XDB-C18 5 μm 9.5×250 mm column) using a linear 1%A→B/min gradient. Linear CuSar-octreotate eluted at 30 min (determinedby MS) and fractions containing linear CuSar-octreotate werelyophilised. The dried fractions were then redissolved in 25 mM ammoniumacetate (8 mL) and an excess of 2,2-dithiodipyridine (12 mg) was addedto form an intramolecular disulfide bond, cyclizing the octreotate. Thissolution was then applied to a semi-preparative reverse phase HPLCcolumn, and purified using a linear 1% A→B/min gradient. CyclicCuSar-octreotate eluted at 31.5 min and fractions containing cyclicCuSar-octreotate were lyophilised to give a light blue pellets.CuSar-NHCO(CH₂)₃CO-octreotate: 4 mg; HPLC retention time: 12.049 min(linear gradient, 0→60% B in A over 25 min). MS: [CuC₆₈H₁₀₂N₁₈O₁₄S₂]²⁺:760.8296 (experimental), 760.8280 (calculated); [CuC₆₈H₁₀₁N₁₈O₁₄S₂]²⁺:1520.6513 (experimental), 1520.6482 (calculated).

Example 8 Sar-Lys₃-BBN: (Also Called L¹-Lys₃-BBN)

Bombesin peptide (BBN) (1-14)(Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (Seq IDNo:2)) was synthesised on Fmoc-PAL-PEG-PS resin using standard Fmocsolid phase peptide synthesis procedures. The side chain of the Lyssresidue was protected with a iv-Dde(Nε-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) group. This wasselectively deprotected using 5% hydrazine in DMF (3×5 mL) over onehour. Resin (0.05 g, ˜0.2 mmol/g) was swelled in DMF. L¹-(t-Boc)₃₋₅ (10mg, 0.012 mmol), HATU (10 mg, 0.026 mmol) and diisopropylethylamine (20μL) in DMF (1 mL) were added to the resin and the mixture was stirredand left to react for 3 hrs. The reaction supernatant was then drainedand the resin washed with DMF (3×5 mL) and DCM (3×5 mL). The resin wastransferred to a falcon tube and trifluoroacetic acid (2 mL), deionisedwater (70 μL) and triisopropylsilane (70 μL) were added. This solutionwas placed on a shaker for 40 min. The solvent was evaporated under astream of N₂ gas and the residue dissolved in A:B (70:30). This solutionwas filtered (MilliQ 0.45 m syringe filter) and lyophilised.

The crude peptide material was purified by semi-preparative reversephase HPLC (Phenomenex Synergi 4u Hydro-RP 80 A 50×21.20 mm), using a“slow” linear gradient (0.5% A→B/min). L¹-Lys₃-bombesin eluted with 25%B in A and fractions containing L¹-Lys₃-bombesin were lyophilised. Thepeptide was not pure, so the dried fractions were redissolved in milliQwater and applied to the same semi-preparative reverse phase HPLCcolumn, and purified using a “very slow” linear 0.25% A→B/min gradient.L¹-Lys₃-bombesin eluted with ˜26.5% B in A. An impurity with a lowermolecular mass (˜780) still persisted in these fractions, so the peptidewas purified by semi-preparative reverse HPLC (Eclipse XDB-C18 5 μm9.5×250 mm column) using a using a “slow” linear gradient (0.5%A→B/min). L¹-Lys₃-bombesin eluted with 26% B in A and fractionscontaining L¹-Lys₃-bombesin were lyophilised. Analytical reverse phaseHPLC indicated that the final fractions contained 95% L¹-Lys₃-bombesin.L¹-Lys₃-bombesin: 1-50 μg; HPLC retention time: 13.59 min (lineargradient, 0→60% B in A over 25 min); MS: [C₉₀H₁₅₂N₃₀O₂₀S₂]⁴⁺: 501.54(experimental), 501.54 (calculated); [C₉₀H₁₅₁N₃₀O₂₀S₂]³⁺: 668.39(experimental), 668.38 (calculated); [C₉₀H₁₅₀N₃₀O₂₀S₂]²⁺: 1002.07(experimental), 1002.07 (calculated).

Example 9 L¹-Tyr

Fmoc-Tyr(tBu)-OH was coupled to 2-chlorotrityl resin (˜1.3 mmol/g) usingstandard coupling procedures. The Fmoc protecting group was removedusing 50% piperidine in DMF and the resin was washed with DMF (3×5 mL).Tyr resin (0.08 g) was swelled in DMF (0.5 mL). L¹-(t-Boc)₃₋₅ (12 mg,0.014 mmol), HATU (10 mg, 0.026 mmol) and diisopropylethylamine (10 μL)in DMF (1 mL) were added to the resin and the mixture was stirred andleft to react for 2 hrs. The reaction supernatant was then drained andthe resin washed with DMF (3×5 mL) and DCM (3×5 mL). The resin wastransferred to a falcon tube and trifluoroacetic acid (2 mL), deionisedwater (70 μL) and triisopropylsilane (70 μL) were added. This solutionwas placed on a shaker for 40 min. The solvent was evaporated under astream of N₂ gas and the residue dissolved in A:B (70:30). This solutionwas filtered (MilliQ 0.45 μm syringe filter) and lyophilised.

The crude peptide material was purified by semi-preparative reversephase HPLC (Eclipse XDB-C18 5 μm 9.5×250 mm column) using a linear 1%A→B/min gradient. L¹-Tyr eluted with 13% B in A (determined by ESI-MS)and fractions containing L¹-Tyr were lyophilised. L¹-Tyr: ˜1 mg. MS:[C₂₈H₅₁N₉O₅]²⁺296.72 (experimental), 296.70 (calculated);[C₂₈H₅₀N₉O₅]⁺592.40 (experimental), 592.39 (calculated). HPLC: 7.360 min(linear gradient, 0→60% B in A over 25 min). ¹H NMR: δ 1.76, m, 2H,glutarate CH₂; 2.13, m, 2H, glutarate CH₂; 2.24, t, 2H glutarate CH₂;2.92, m, 1H, tyrosine β CH₂; 3.20-3.35, tyrosine β CH₂ obscured by cagesignals; 3.20, broad s, 6H, cage CH₂; 3.26, broad s, 6H, cage CH₂; 3.35,broad s, 6H, cage CH₂; 3.53, broad s, 6H cage CH₂; 4.61, m, 1H, tyrosinea CH; 6.88, d, 2H, tyrosine aromatic CH; 7.12, d, 2H, tyrosine aromaticCH.

Example 10 L²-(YaHxC)₂

The synthesis of this molecule is as shown below:

Tyr(tBu)-aHx-Cys(Trt)-NH₂ was synthesised on Nova Peg Rink Amide resinwith a loading of 0.67 mmol g⁻¹. HATU (8.9 mg, 0.02 mmol, 2 equivalents)in DMF (0.5 mL) and DIPEA (7 L) was added to a solution of L²-(t-Boc)₄(10 mg, 0.01 mmol) in DMF (0.5 mL). This solution was added toTyr(tBu)-aHx-Cys(Trt)-NH₂ on resin (0.05 g, ˜0.22 mmol g⁻¹, ˜0.01 mmol)and the mixture was left to react overnight. The reaction supernatantwas then drained and the resin washed with DMF (3×5 mL) and DCM (3×5mL). The resin was transferred to a falcon tube and trifluoroacetic acid(3 mL), deionised water (80 μL) and triisopropylsilane (80 u mL) wereadded. The falcon tube was placed on a shaker for 90 min. After thistime, the solvent was evaporated under a stream of nitrogen gas, and theresidue redissolved in A:B (50:50). This solution was filtered (MilliQ0.45 μm syringe filter) and lyophilised.

The crude peptide material was purified by semi-preparative reversephase HPLC (Eclipse XDB-C18 5 μm 9.5×250 mm column) using a linear 1%A→B/min gradient. L²-(YHxC)₂ eluted at 27 min (determined by ESI-MS).Fractions containing L²-(YHxC)₂ were lyophilised. This sample wassubjected to a second HPLC purification where it eluted at 44 min usinga linear 0.5% A→B/min gradient. L²-(YHxC)₂: ˜ 1 mg; HPLC retention time:10.52 min (linear gradient, 0→60% B in A over 25 min) MS:[C₆₀H₁₀₀N₁₆O₁₂S]²⁺650.36 (calculated), 650.36 (experimental);[C₆₀H₉₉N₁₆O₁₂S]⁺1299.71 (calculated), 1299.71 (experimental)¹H NMR: δ1.16, m, 4H, CH₂ (f); 1.38, m, 4H, CH₂ (e); 1.59, m, 4H, CH₂ (g); 1.79,m, 4H, CH₂ (b); 2.13, m, 4H, CH₂ (a); 2.29, t, 4H, CH₂ (c); 2.34, t, 4H,CH₂ (h); 2.29-3.08, m, 8H, βCH₂ Tyr and Cys; 3.05, m, 2H, CH (d); 3.17,m, 2H, CH (d); 3.17, s, 12H, cage CH₂; 3.38, s, 12H, cage CH₂;4.44-4.55, m, 4H, αCH Tyr and Cys; 6.89, d, 4H, Tyr ar; 7.18, d, 4H, Tyrar;

Example 11 L²-(YaHxC)

L²-(YaHxC) was synthesised in the same way and on the same scale as L².(YaHxC)₂, except that instead of HATU, diisopropylcarbodiimide (7 μL)and hydroxybenzotriazole (3.5 mg) were used as coupling agents. A schemeis shown below.

A mass spectrum of the crude peptide material indicated that bothL²-(YaHxC)₂ and L²-(YaHxC) were present. The crude peptide material waspurified by semi-preparative reverse phase HPLC (Eclipse XDB-C18 5 μm9.5×250 mm column) using a linear 1% A→B/min gradient. L²-(YHxC) elutedat 23.5 min and L²-(YaHxC)₂ eluted at 28.5 min (determined by ESI-MS).Fractions containing L²-(YHxC) were lyophilised. This sample wassubjected to a second HPLC purification where it eluted at 35.8 minusing a linear 0.5% A→B/min gradient. L²-(YaHxC): ˜0.2-0.6 mg; HPLCretention time: 8.73 min (linear gradient, 0→60% B in A over 25 min) MS:[C₄₂H₇₄N₁₂O₉S]²⁺ 461.27 (experimental), 461.27 (calculated);[C₄₂H₇₃N₁₂O₉S]⁺921.53 (calculated), 921.53 (experimental). ¹H NMR: δ1.15, m, 2H, CH₂ (i); 1.37, m, 2H, CH₂ (h); 1.58, m, 2H, CH₂ (j); 1.79,m, 2H, CH₂ (e); 1.89, m, 2H, CH₂ (b); 2.16, m, 2H, CH₂ (d); 2.30, t, 2H,CH₂ (f); 2.34, t, 2H, CH₂ (k); 2.37, t, 4H, CH₂ (a) and CH₂ (c);2.87-3.03, m, 4H, βCH₂ Tyr and Cys; 3.03, m, 1H, CH (g); 3.22, m, 1H, CH(g); 3.23, s, 12H, cage CH₂; 3.42, s, 6H, cage CH₂; 3.50, s, 6H, cageCH₂; 4.44-4.55, m, 2H, αCH Tyr and Cys; 6.89, d, 2H, Tyr ar; 7.18, d,2H, Tyr ar;

Example 12 c(RGDfK(maleimidopropionate))

D(tBu)fK(ivDde)R(Pbf)G-OH (1 mmol) was synthesised on chlorotrityl resin(1 mmol g⁻¹, 1 g). The N-terminus was protected with trityl chloride togive Trt-D(tBu)fK(ivDde)R(Pbf)G-OH, followed by removal of the ivDdeprotecting group of the Lys side-chain using hydrazine (3×5 mL 5%hydrazine in DMF). Maleimidopropionic acid was then coupled to the Lysside-chain to give Trt-D(tBu)fK(maleimidopropionate)R(Pbf)G-OH. Thisspecies was concurrently deprotected at the N-terminus and cleaved fromthe resin using 2% TFA in DCM with radical scavengers to giveD(tBu)fK(maleimidopropionate)R(Pbf)G-OH. The resulting solution wasfiltered and the solvent removed under reduced pressure, before HCTU (1equivalent) and DIPEA (1 mL) in DCM (5 mL) were added to cyclise thepeptide. The solution was stirred for 2 hours and after this time thesolvent was removed under reduced pressure. The remaining protectinggroups were then removed with TFA:H₂O:TIPS (95:2.5:2.5). The TFA wasevaporated under a scream of nitrogen gas, and the crude material wasredissolved in A:B (50:50), filtered and lyophilised. The crude peptidematerial was purified by semi-preparative reverse phase HPLC (EclipseXDB-C18 5 μm 9.5×250 mm column) using a linear 1% A→B/min gradient.c(RGDfK(maleimidopropionate)) eluted at 28.5 min.c(RGDfK(maleimidopropionate)): 1-2 mg; HPLC retention time: 15.31 min(linear gradient, 0→60% B in A over 25 min) MS: [C₃₄H₄₆N₁₀O₁₀]⁺755.35(experimental), 755.35 (calculated).

Example 13 L²-(RGD)₂

This molecule is synthesized as shown in the attached scheme:

L²-(YaHzC)₂ (˜0.5 mg) and c(RGDfK(maleimidopropionate)) (˜1 mg) weredissolved in ammonium acetate solution (25 mM, pH=6.5). After sitting atroom temperature for ˜30 min, the solution was purified bysemi-preparative reverse phase HPLC (Eclipse XDB-C18 5 μm 9.5×250 mmcolumn) using a linear 1% A→B/min gradient.c(RGDfK(maleimidopropionate)) eluted at 26 min and L²-(RGD)₂ eluted at29 min. L²-(RGD)₂: L²-(RGD)₂: ˜ 1 mg; HPLC retention time: 16.54 min(linear gradient, 0→60% B in A over 25 min) MS:[C₁₂₈H₁₉₄N₃₆O₃₂S₂]⁴⁺703.11 (experimental), 703.10 (calculated);[C₁₂₈H₁₉₃N₃₆O₃₂S₂]³⁺937.14 (experimental), 937.13 (calculated).

Example 14 HPLC-MS of Sar-Peptides

HPLC-MS traces were acquired for Sar-peptides synthesised. HPLC-MStraces were also acquired for solutions containing Sar-peptide andCuCl₂, to determine whether Sar-peptide binds “free” Cu²⁺ ion. We used areverse phase C18 analytical HPLC column with a linear gradient (0→60% Bin A over 25 min) to determine retention times and molecular masses ofpeptide species. (Here, A=milliQ water with 0.1% formic acid;B=acetonitrile with 0.1% formic acid.)

L¹-Tyr: L¹-Tyr (˜10 μg) was dissolved in milliQ water (50 μL). Thissolution was further diluted (5 μL of peptide solution in 10 μL ofmilliQ water) to provide a solution of suitable concentration forHPLC-MS. A solution containing L¹-Tyr (5 μL of the original stockpeptide solution) and CuCl₂ (10 μL of 1 mM CuCl₂ solution) in milliQwater (15 μL) was also made up. LCMS: L¹-Tyr, R.T.: 5.992;[C₂₈H₅₁N₉O₅]²⁺296.72 (experimental), 296.70 (calculated);[C₂₈H₅₀N₉O₅]⁺592.40 (experimental), 592.39 (calculated);[Cu(L¹-Tyr)]^(x+), R.T.: 6.485; [CuC₂₈H₄₉N₉O₅]²⁺327.17 (experimental),327.16 (calculated), [CuC₂₈H₄₈N₉O₅]⁺653.31 (experimental), 653.31(calculated).

L¹-Tyr³-octreotate: L¹-Tyr³-octreotate (˜10 μg) was dissolved in milliQwater (50 μL). This solution was further diluted (5 μL of peptidesolution in 20 μL of milliQ water) to provide a solution of suitableconcentration for HPLC-ESI-MS. A solution containing L¹-Tyr³-octreotate(5 μL of the original stock peptide solution) and CuCl₂ (5 μL of 1 mMCuCl₂ solution) in milliQ water (15 μL) was also made up. HPLC-ESI-MS:L¹-Tyr³-octreotate, R.T.: 12.984 min; [C₆₈H₁₀₆N₁₈O₁₄S₂]⁴⁺365.69(experimental), 365.69 (calculated); [C₆₈H₁₀₅N₁₈O₁₄S₂]³⁺487.25(experimental), 487.25 (calculated); [C₆₈H₁₀₄N₁₈O₁₄S₂]²⁺730.37(calculated), 730.37 (experimental); [Cu(L¹-Tyr³-octreotate)]^(x+),R.T.: 13.199 min; [CuC₆₈H₁₀₄N₁₈O₁₄S₂]⁴⁺380.92 (experimental), 380.92(calculated); [CuC₆₈H₁₀₃N₁₈O₁₄S₂]³⁺507.56 (experimental), 507.55(calculated); [CuC₆₈H₁₀₂N₁₈O₁₄S₂]²⁺760.83 (calculated), 760.83(experimental).

(L¹)₂-Tyr³-octreotate: (L¹)₂-Tyr³-octreotate (˜10 μg) was dissolved inmilliQ water (50 μL). This solution was further diluted (5 μL of peptidesolution in 20 μL of milliQ water) to provide a solution of suitableconcentration for HPLC-MS. A solution containing (L¹)₂-Tyr³-octreotate(5 μL of the original stock peptide solution) and CuCl₂ (5 μL of 1 mMCuCl₂ solution) in milliQ water (15 μL) was also made up. LCMS:(L¹)₂-Tyr³-octreotate, R.T.: 11.213; [C₈₇H₁₄₅N₂₆O₁₆S₂]⁵⁺375.02(experimental), 375.02 (calculated); [C₈₇H₁₄₄N₂₆O₁₆S₂]⁴⁺468.52(experimental), 468.52 (calculated); [C₈₇H₁₄₃N₂₆O₁₆S₂]³⁺624.36(experimental), 624.36 (calculated); [C₈₇H₁₄₂N₂₆O₁₆S₂]²⁺936.03(experimental), 936.03 (calculated); [Cu₂(L¹)₂-Tyr³-octreotate]^(x)+,R.T.: 11.938; [Cu₂C₈₇H₁₄₁N₂₆O₁₆S₂]⁵⁺399.58 (experimental), 399.58(calculated); [Cu₂C₈₇H₁₄₀N₂₆O₁₆S₂]⁴⁺ 499.23 (experimental), 499.23(calculated); [Cu₂C₈₇H₁₃₉N₂₆O₁₆S₂]³⁺665.30 (experimental), 665.29(calculated).

L¹-Lys₃-bombesin: L¹-Lys₃-bombesin ((˜1-5 μg) was dissolved in milliQwater (50 μL). The concentration of this solution was suitable forHPLC-ESI-MS. 1 mM CuCl₂ solution (2.5 μL) was added to a portion of thissolution (20 μL) to determine whether L¹-Lys₃-bombesin binds “free” Cu²⁺ion. HPLC-ESI-MS: L¹-Lys₃-bombesin, R.T.: 13.520 min; [C₉₀H₁₅₂N₃₀O₂₀S]⁴⁺: 501.54 (experimental), 501.54 (calculated); [C₉₀H₁₅₁N₃₀O₂₀S]³⁺: 668.39(experimental), 668.38 (calculated); [C₉₀H₁₅₀N₃₀O₂₀S₂]²⁺: 1002.07(experimental), 1002.07 (calculated); [Cu(L1-Lys₃-bombesin)]^(x)+, R.T.:13.769 min; [CuC₉₀H₁₅₀N₃₀O₂₀S]⁴⁺ : 517.02 (experimental), 517.02(calculated); [CuC₉₀H₁₄₉N₃₀O₂₀S]³⁺: 689.03 (experimental), 689.02(calculated); [CuC₉₀H₁₄₈N₃₀O₂₀S]²⁺: 1033.03 (experimental), 1033.03(calculated).

Example 15 Radiolabelling with ⁶⁴Cu

⁶⁴CuCl₂ (1.88 GBq/mL, 0.1 M HCl pH 1) was purchased from ANSTOradiopharmaceuticals and industrials (ARI), Lucas Heights, NSW,Australia. The radionuclidic purity at calibration {(⁶⁴Cu)/(⁶⁷Cu)} was100% and the radiochemical purity as Cu²⁺ was 100%. The chemical purityof copper, zinc and iron were 1.1 μg/mL, 0.9 μg/mL and 10 μg/mLrespectively.

HPLC with a radioactivity sodium iodide scintillation detector was usedto monitor L¹-peptide ligation to ⁶⁴Cu²⁺. We employed a reverse phaseC18 analytical HPLC column with a linear gradient (0→60% B in A over 15min) to determine retention times of [⁶⁴CuL¹-peptides]²⁺. Theseretention times were compared with retention times of the “cold”[CuL¹-peptides]²⁺ under the same HPLC conditions with UV spectroscopicdetection at 275 nm.

[⁶⁴Cu(L¹)]²⁺: Trace 1: ⁶⁴CuCl₂ (38 MBq, 20 μL, 0.1 M HCl) was added toan aqueous solution (580 μL) containing L¹ (0.015 mg/mL) and sodiumacetate (0.015 M). The pH was ˜4.5 (measured with pH strips). Thesolution was left at ambient temperature for 10 min before an aliquot(100 μL) was injected onto a reverse phase C18 analytical HPLC columnwith a linear gradient (0→60% B in A over 15 min). Retention time: 2.33min, ˜82% radiochemical yield. Retention time: 8.36, ˜ 18% radiochemicalyield. Trace 2: Sodium acetate solution (100 μL, 0.1 M) was added to theremainder of the above solution. The pH was ≥5.5. An aliquot of thissolution (100 μL) was injected onto a reverse phase C18 analytical HPLCcolumn with a linear gradient (0→60% B in A over 15 min). Retentiontime: 8.334 min, >95% radiochemical yield. Trace 3: ⁶⁴CuCl₂ (38 MBq, 20μL, 0.1 M HCl) was added to the remainder of the above solution toincrease the signal to noise ratio, as well as determine the timerequired to complex 100% of ⁶⁴Cu²⁺ present in solution. A furtheraliquot of sodium acetate (100 μL, 0.1 M) solution was added. Thesolution was left to stand at ambient temperature for 5 min before analiquot was injected onto a reverse phase C18 analytical HPLC columnwith a linear gradient (0→60% B in A over 15 min). Retention time: 8.334min, >95% radiochemical yield. An aqueous sample of “cold”[Cu(L¹)](NO₃)₃ (˜1 mg/mL) was injected onto the HPLC column (injectionvolume ˜30 μL) and was eluted using the same linear gradient (0→60% B inA over 15 min). Retention time: 8.573 min.

[⁶⁴Cu(L¹-Tyr)]²⁺: ⁶⁴CuCl₂ (19 MBq, 10 μL, 0.1 M HCl) was added to anaqueous solution (490 μL) containing L¹-Tyr (0.02 mg/mL) and sodiumacetate (0.02 M). The solution was left at ambient temperature for 10min before an aliquot (100 μL) was injected onto a reverse phase C18analytical HPLC column with a linear gradient (0→60% B in A over 15min). Retention time: 10.231 min, >95% radiochemical yield. An aqueoussample of “cold” [Cu(L¹-Tyr)]²⁺: (˜1 mg/mL) was injected onto the HPLCcolumn (injection volume ˜30 μL) and was eluted using the same lineargradient (0→60% B in A over 15 min). Retention time: 10.403 min

[⁶⁴CuL¹-Tyr³-octreotate]²⁺: An aliquot of 0.1 M HCl solution containing⁶⁴CuCl₂ (38 MBq, 20 μL, 0.1 M HCl) was added to an aqueous solution (490μL) containing L¹-Tyr³-octreotate (0.02 mg/mL) and sodium acetate (0.02M). The pH was >5.5. The solution was left at ambient temperature for 20min before an aliquot (100 μL) was injected onto a reverse phase C18analytical HPLC column with a linear gradient (0→60% B in A over 15min). Retention time: 12.730 min, >95% radiochemical yield. An aqueoussample of “cold” [CuL¹-Tyr³-octreotate]²⁺ (˜1 mg/mL) was injected ontothe HPLC column (injection volume ˜30 μL) and was eluted using the samelinear gradient (0→60% B in A over 15 min). Retention time: 12.937 min.

Human male AB serum (200 μL) was added to a solution of[⁶⁴CuL¹-Tyr³-octreotate]²⁺ (200 μL). This solution was incubated in awater bath at 37° C. At time points of 1 hr, 4 hr and 20 hr an aliquotof this serum solution (100 μL) was removed for radio-HPLC analysis:acetonitrile (200 μL) was added to the serum aliquot to precipitateserum proteins. This mixture was filtered and the acetonitrileevaporated under a stream of argon gas. The final volume was <100 μL.The solution was frozen at −70° C. until injection onto the HPLC column.1 hr: Retention time 12.716 min, >95% radiochemical yield; 4 hr:Retention time 12.725 min, >95% radiochemical yield; 20 hr: Retentiontime 12.800 min, >95% radiochemical yield.

[⁶⁴CuL¹-Lys₃-bombesin]²⁺: An aliquot of 0.1 M HCl solution containing⁶⁴CuCl₂ (38 MBq, 20 μL, 0.1 M HCl) was added to an aqueous solution (290μL) containing L¹-Lys₃-bombesin (˜0.03 mg/mL) and sodium acetate (0.03M). The solution was left at ambient temperature for 10 min before analiquot (100 μL) was injected onto a reverse phase C18 analytical HPLCcolumn with a linear gradient (0→60% B in A over 15 min). Retentiontime: 12.642; >95% radiochemical yield. An aqueous sample of “cold”

[CuL¹-Lys₃-bombesin]²⁺ (˜0.5 mg/mL) was injected onto the HPLC column(injection volume ˜60 μL) and was eluted using the same linear gradient(0→60% B in A over 15 min). Retention time: 13.073 min.

A single time point serum stability study for [⁶⁴CuL¹-Lys₃-bombesin]²⁺was conducted in the same way as studies for [⁶⁴CuL¹-Tyr³-octreotate]2+.An aliquot of [⁶⁴CuL¹-Lys₃-bombesin]²⁺ (200 μL) was added to human maleAB serum and incubated at 37° C. After 2 hr, the sample was subjected tothe same treatment as described above and injected onto the HPLC column.2 hr: Retention time 12.828 min, >95% radiochemical yield.

Finally, it will be appreciated that various modifications andvariations of the methods and compositions of the invention describedherein will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are apparent tothose skilled in the art are intended to be within the scope of thepresent invention.

1. A compound of formula (I),

wherein: L is a nitrogen containing macrocyclic metal ligand; X is alinking moiety; Y is selected from the group consisting of OR, SR¹ andN(R²)₂; R is independently selected from the group consisting of H, anoxygen protecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl; R¹ is independently selectedfrom the group consisting of H, a sulfur protecting group, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl; and each R² is independently selected from the groupconsisting of H, a nitrogen protecting group, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionallysubstituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl;or a pharmaceutically acceptable salt or complex thereof.
 2. Thecompound according to claim 1 wherein X is a linking moiety having from1 to 20 atoms in the normal chain.
 3. The compound according to claim 1wherein X is a group of the formula:—(CH₂)_(q)CO(AA)_(r)NH(CH₂)_(s)— wherein each AA is independently anamino acid group; q is an integer selected from the group consisting of1, 2, 3, 4, 5, 6, 7, and 8; r is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; and s is an integerselected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 4.The compound according to claim 3 wherein q is 3, r is 0 and s is
 5. 5.The compound according to claim 1 wherein X is a group of the formula—(CH₂)_(n)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR³ where R³ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; and n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 6. The compound according to claim5 wherein n is an integer selected from the group consisting of 1, 2, 3,4, and
 5. 7. The compound according to claim 5 wherein X is selectedfrom the group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.
 8. The compound according to claim 5wherein X is —(CH₂)₃—.
 9. The compound according to claim 1 wherein Y isOH.
 10. The compound according to claim 1 wherein L is a nitrogencontaining cage metal ligand.
 11. The compound according to claim 10wherein L is a nitrogen containing cage metal ligand of the formula:

wherein V is selected from the group consisting of N and CR⁴; each R^(x)and R^(y) are independently selected from the group consisting of H,CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO; each p isindependently an integer selected from the group consisting of 2, 3, and4; and R⁴ is selected from the group consisting of H, OH, halogen, NO₂,NH₂, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₆-C₁₈aryl, cyano, CO₂R⁵, NHR⁵, N(R⁵)₂ and a group of the formula:

wherein X¹ is a linking moiety; Y¹ is selected from the group consistingof OR⁶, SR⁷, N(R⁸)₂ and a molecular recognition moiety; wherein R⁵ is Hor C₁-C₁₂alkyl; R⁶ is selected from the group consisting of H, halogen,an oxygen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl; R⁷ isselected from the group consisting of H, halogen, a sulfur protectinggroup, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl and optionallysubstituted C₂-C₁₂ heteroalkyl; and each R⁸ is independently selectedfrom the group consisting of H, a nitrogen protecting group, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl.
 12. The compound according to claim 11 wherein L is amacrocyclic metal ligand of the formula:

wherein each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHOand each p is independently an integer selected from the groupconsisting of 2, 3, and
 4. 13. The compound according to claim 1 whereinL is a macrocyclic ligand of the formula:

wherein each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHOand each p is independently an integer selected from the groupconsisting of 2, 3, and
 4. 14. The compound according to claim 1 whereinL is selected from the group consisting of:

wherein R⁴ is selected from the group consisting of H, OH, halogen, NO₂,NH₂, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₆-C₁₈aryl, cyano, CO₂R⁵, NHR⁵, N(R⁵)₂ and a group of the formula:

wherein X¹ is a linking moiety; and Y¹ is selected from the groupconsisting of OR⁶, SR⁷, N(R⁸)₂ and a molecular recognition moiety. 15.The compound according to claim 11, wherein R⁴ is selected from thegroup consisting of NH₂, CH₃ and a group of the formula:


16. The compound according to claim 15 wherein X¹ is a linking moietyhaving from 1 to 20 atoms in the normal chain.
 17. The compoundaccording to claim 15 wherein X¹ is a group of the formula:—(CH₂)_(t)CO(AA)_(u)NH(CH₂)_(v)— wherein each AA is independently anamino acid group; t is an integer selected from the group consisting of1, 2, 3, 4, 5, 6, 7, and 8; u is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; and v is an integerselected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 18.The compound according to claim 17 wherein t is 3, u is 0 and v is 5.19. The compound according to claim 15 wherein X¹ is a group of theformula:—(CH₂)_(a)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR⁹ where R⁹ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; and a is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 20. The compound according to claim19 wherein a is an integer selected from the group consisting of 1, 2,3, 4, and
 5. 21. The compound according to claim 19 wherein X isselected from the group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.
 22. The compound according to claim 19wherein X¹ is —(CH₂)₃—.
 23. The compound according to claim 15 whereinY¹ is OH.
 24. The compound according to claim 1 wherein L is a group ofthe formula:


25. The compound according to claim 1 wherein the compound is selectedfrom the group consisting of:


26. A compound of formula (II):

wherein: L is a nitrogen containing macrocyclic metal ligand; X is alinking moiety; and Y is a molecular recognition moiety; or apharmaceutically acceptable salt or complex thereof.
 27. The compoundaccording to claim 26 wherein X is a linking moiety having from 1 to 20atoms in the normal chain.
 28. The compound according to claim 26wherein X is a group of the formula:—(CH₂)_(q)CO(AA)_(r)NH(CH₂)_(s)— wherein each AA is independently anamino acid group; q is an integer selected from the group consisting of0, 1, 2, 3, 4, 5, 6, 7, and 8; r is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; and s is an integerselected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 29.The compound according to claim 28 wherein r is 0 and s is
 5. 30. Thecompound according to claim 26 wherein X is a group of the formula—(CH₂)_(n)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR³ where R³ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; and n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 31. The compound according to claim30 wherein n is an integer selected from the group consisting of 1, 2,3, 4, and
 5. 32. The compound according to claim 30 wherein X isselected from the group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.
 33. The compound according to claim 30wherein X is —(CH₂)₃—.
 34. The compound according to claim 26 wherein Lis a nitrogen containing cage metal ligand.
 35. The compound accordingto claim 34 wherein L is a nitrogen containing cage metal ligand of theformula:

wherein V is selected from the group consisting of N and CR⁴; each R^(x)and R^(y) are independently selected from the group consisting of H,CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO; each p isindependently an integer selected from the group consisting of 2, 3, and4; and R⁴ is selected from the group consisting of H, OH, halogen, NO₂,NH₂, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₆-C₁₈aryl, cyano, CO₂R⁵, NHR⁵, N(R⁵)₂ and a group of the formula:

wherein X¹ is a linking moiety; Y¹ is selected from the group consistingof OR⁶, SR⁷, N(R⁸)₂ and a molecular recognition moiety; wherein R⁵ is Hor C₁-C₁₂alkyl; R⁶ is selected from the group consisting of H, halogen,an oxygen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl; R⁷ isselected from the group consisting of H, halogen, a sulfur protectinggroup, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl and optionallysubstituted C₂-C₁₂ heteroalkyl; and each R⁸ is independently selectedfrom the group consisting of H, a nitrogen protecting group, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl.
 36. The compound according to claim 26 wherein L is amacrocyclic metal ligand of the formula:

wherein each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO;and each p is independently an integer selected from the groupconsisting of 2, 3, and
 4. 37. The compound according to claim 26wherein L is a macrocyclic ligand of the formula:

wherein each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO;and each p is independently an integer selected from the groupconsisting of 2, 3, and
 4. 38. The compound according to claim 26wherein L is selected from the group consisting of:

wherein R⁴ is selected from the group consisting of H, OH, halogen, NO₂,NH₂, optionally substituted C₁-C₁₂alkyl, optionally substitutedC₆-C₁₈aryl, cyano, CO₂R⁵, NHR⁵, N(R⁵)₂ and a group of the formula:

wherein X¹ is a linking moiety; and Y¹ is selected from the groupconsisting of OR⁶, SR⁷, N(R⁸)₂ and a molecular recognition moiety. 39.The compound according to claim 35, wherein R⁴ is selected from thegroup consisting of NH₂, CH₃ and a group of the formula:

wherein X¹ is a linking moiety; Y¹ is selected from the group consistingof OR⁶, SR⁷, N(R⁸)₂ and a molecular recognition moiety; R⁶ is selectedfrom the group consisting of H, halogen, an oxygen protecting group,optionally substituted C₁-C₁₂alkyl, optionally substitutedC₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl and optionallysubstituted C₂-C₁₂ heteroalkyl; R⁷ is selected from the group consistingof H, halogen, a sulfur protecting group, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionallysubstituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl;and each R⁸ is independently selected from the group consisting of H, anitrogen protecting group, optionally substituted C₁-C₁₂alkyl,optionally substituted C₂-C₁₂alkenyl, optionally substitutedC₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl.
 40. Thecompound according to claim 39 wherein X¹ is a linking moiety havingfrom 1 to 20 atoms in the normal chain.
 41. The compound according toclaim 15 wherein X¹ is a group of the formula:—(CH₂)_(t)CO(AA)_(u)NH(CH₂)_(v)— wherein each AA is independently anamino acid group; t is an integer selected from the group consisting of1, 2, 3, 4, 5, 6, 7, and 8; u is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; and v is an integerselected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and
 8. 42.The compound according to claim 41 wherein t is 3, u is 0 and v is 5.43. The compound according to claim 39 wherein X¹ is a group of theformula—(CH₂)_(a)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR⁹ where R⁹ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; and a is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 44. The compound according to claim43 wherein a is an integer selected from the group consisting of 1, 2,3, 4, and
 5. 45. The compound according to claim 43 wherein X isselected from the group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.
 46. The compound according to claim 43wherein X¹ is —(CH₂)₃—.
 47. The compound according to claim 39 whereinY¹ is OH.
 48. The compound according to claim 26 wherein L is a group ofthe formula:


49. The compound according to claim 39 wherein Y¹ is a molecularrecognition moiety.
 50. The compound according to claim 26 wherein eachmolecular recognition moiety includes a spacer portion and a recognitionportion, wherein the spacer portion joins the recognition portion to theremainder of the molecule.
 51. The compound according to claim 26wherein the molecular recognition moiety or recognition portion isselected from the group consisting of an antibody, a protein, a peptide,a carbohydrate, a nucleic acid, an oligonucleotide, an oligosaccharideand a liposome or a fragment or derivative thereof.
 52. The compoundaccording to claim 51 wherein the molecular recognition moiety ormolecular recognition portion is a peptide or a fragment or a derivativethereof.
 53. The compound according to claim 52 wherein the molecularrecognition moiety or molecular recognition portion is selected from thegroup consisting of [Tyr³]-octreotate, Lys³-bombesin and cyclic RGD. 54.The compound according to claim 1, wherein the nitrogen containingmacrocyclic metal ligand is coordinated with a metal ion.
 55. Thecompound according to claim 54 wherein the metal ion is a radionuclideselected from the group consisting of Cu, Tc, Gd, Ga, In, Co, Re, Fe,Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg, andTi.
 56. The compound according to claim 54, wherein the metal ion is aradionuclide selected from the group consisting of ⁶⁰Cu, ⁶²Cu, ⁶⁴Cu and⁶⁷Cu.
 57. A pharmaceutical composition comprising the compound accordingto claim
 1. 58. A method of producing a compound of formula (I) asclaimed in claim 1:

the method comprising: (a) reacting an amino substituted metal chelatingligand or a metal complex thereof of the formula:L-NH₂ wherein L is a nitrogen containing macrocyclic metal ligand; withan activated dicarbonyl compound; and (b) isolating the compound offormula (I) or a metal complex thereof.
 59. The method according toclaim 58 wherein the activated dicarbonyl compound is a compound of theformula (III):

wherein Z is O, S or NR².
 60. The method according to claim 58 whereinthe activated dicarbonyl compound is a compound of the formula (IV):

wherein Lv is a leaving group.
 61. The method according to claim 59wherein the leaving group is selected from the group consisting of Cl,Br, CH₃SO₃, CH₃C₆H₄SO₃, and a group of the formula:


62. The method according to claim 58 wherein the amino substituted metalchelating ligand and the activated dicarbonyl compound are reacted inthe presence of a base.
 63. The method according to claim 62, whereinthe base is diisopropylethylamine.
 64. The method according to claim 58wherein L is a nitrogen containing cage metal ligand.
 65. A method oftreating or preventing a condition in a subject, the method comprisingadministering a therapeutically effective amount of the compoundaccording to claim 54 to a subject.
 66. The method according to claim65, wherein the condition is cancer.
 67. A method of radioimaging asubject, the method comprising administering an effective amount of thecompound according to claim 54 to the subject.